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What is the role of triton in buffer for western blot assay?

What is the role of triton in buffer for western blot assay?



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What is the role of triton in buffer for western blot assay? I understand it has to do with preventing non-specific binding of the antibody.


That's exactly it. Triton is a detergent and incorporating it just makes it a little more difficult for everything to bind. Here's how I imagine blocking buffer working:

PBS/TBS: buffering pH

milk/BSA: uniformly interacting with the entirety of the blot. The idea is that these will a) adsorb to the parts of the blot that don't already have much sample in them (since "empty" areas of the blot would otherwise adsorb the detection antibody/ies (this is a source of nonspecific binding).

Triton (or, more commonly, Tween-20): OK, now think about what's on your blot. Your antibody will have weak interactions with just about anything there. A detergent interacts with everything it touches (antibody and adsorbed proteins on the blot) to reduce binding affinity in lots of these interactions (although, some interesting counterexamples can be found here), thereby making the less-favorable binding even less favorable, decreasing the chances of your detection antibody retaining these weaker (off-target) interactions. The preceding article also suggests that the use of nonionic detergents lowers the signal intensity of the Western (and to a different degree across proteins).


Western Blot Analysis

Leonard G. Davis Ph.D. , . James F. Battey M.D., Ph.D. , in Basic Methods in Molecular Biology , 1986

Publisher Summary

This chapter provides an overview of the western blot analysis method, which allows the investigator to identify specific proteins resolved by SDS polyacrylamide gel electrophoresis by binding with specific antisera. Proteins resolved on an acrylamide gel are transferred to a nitrocellulose (NC) filter that is incubated with the antisera. Transfer buffer used in the western blot analysis method consists of Tris base, glycine, water and methanol. The pH of the transfer buffer is maintained with HCl. The primary antibody specifically binds its epitope and the bound antibody is detected with a secondary species, such as [125] I]-protein A or biotinylated goat anti-IgG. The western blot analysis method involves the preparation of polyacrylamide–SDS gel for separation of proteins, and antibody is obtained against protein sequence of interest. The protein is electrophoretically transferred from the polyacrylamide gel to the NC filter.


Purpose and function of blocking steps

The membrane supports used in western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding.

The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. For example, in applications where AP conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors, including various protein-protein interactions unique to a given set of immunoassay reagents, can influence nonspecific binding. The most important parameter when selecting a blocker is the signal-to-noise ratio, measured as the signal obtained with a sample containing the target analyte, as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background staining and a reduced signal-to-noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the marker enzyme, again causing a reduction of the signal-to-noise ratio. When developing any new immunoassay, it is important to test several different blockers for the highest signal-to-noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.

Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.


What is the role of triton in buffer for western blot assay? - Biology

Here we present a comprehensive review of laboratory detergents and their applications in biomedical experiments. This review includes discussions of ionic, non-ionic and zwitterionic detergents, their general properties as well as information about commonly used detergents from each group. Finally, we include a brief discussion of Labome survey results for some common detergents.

Detergents used in biomedical laboratories are mild surfactants (surface acting agents), used for cell lysis (i.e., the disruption of cell membranes) and the release of intracellular materials. They are amphiphilic molecules, containing both hydrophilic and hydrophobic regions. This amphiphilic property allows detergents to break protein-protein, protein-lipid and lipid-lipid associations, denature proteins and other macromolecules, and prevent nonspecific binding in immunochemical assays and protein crystallization.

There are many types of detergents used in laboratory research. New amphiphilic compounds, usually designed for specific applications, continue to be developed (e.g., maltose-neopentyl glycol [1] and glycosyl-substituted dicarboxylates [2] ). This article reviews the characteristics and applications of the most commonly used laboratory detergents.

TypeChemicals
ionicsodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl
non-ionicTriton X-100, DDM, digitonin, tween 20, tween 80
zwitterionicCHAPS
chaotropicurea

Detergents are amphiphilic organic compounds comprised of a hydrophobic non-polar hydrocarbon moiety (tail) and a hydrophilic polar headgroup (Fig. 1A). This molecular structure is very similar to the amphiphilic phospholipids that make up our cellular membranes, except that the phospholipids possess pair hydrophobic tails attached to the hydrophilic headgroup (Fig 1D). When dissolved in water at appropriate concentrations and temperatures amphiphilic molecules self-assemble into structures that keep their hydrophilic headgroups on the exterior and the hydrophobic tails on the interior away from the water. Due to their molecular differences, detergent molecules form spherical micelles(Fig. 1C) while phospholipids are more likely to develop a bilayer (Fig 1D). The similarity in molecular structures allows the detergent to penetrate phospholipid bilayers and thus disrupt cell membranes.

Furthermore, the hydrophobic core of the micelle can bind to hydrophobic regions of proteins (Fig 1B). The number of detergent molecules in a micelle is called the aggregation number, an important parameter used to assess membrane protein solubility [3]. The length of the hydrophobic region is directly proportional to the degree of hydrophobicity, and it is quite constant among detergents, while the charged headgroup is variable. Both temperature and concentration are important parameters of phase separation and solubility of a detergent. The minimal detergent concentration at which micelles are observed at a given temperature is called the Critical Micelle Concentration (CMC). At any concentrations lower than the CMC, only monomers are observed at concentrations higher than CMC both micelles and monomers co-exist, along with other non-micellar phases that are not dissolved in water. Likewise, the lowest temperature at which micelles are formed is called Critical Micelle Temperature (CMT). CMC is also affected by the degree of lipophilicity of the headgroup. Generally, a low lipophilic or lipophobic character results in high CMC.

Common detergents are categorized into three groups based on their characteristics: ionic (anionic or cationic), non-ionic and zwitterionic. Below I discuss common detergents in each of these categories and provide important information about the selection and use of laboratory detergents.

Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be either anionic or cationic. They generally have higher CMC values than non-ionic detergents and tend to be fairly harsh. Due to their charged headgroups, ionic detergents cannot be removed by ion exchange chromatography. Furthermore, additional precautions should be taken when using ionic detergents because some of their properties may be altered in buffers with variable ionic strength (e.g., CMC can fall dramatically when the NaCl concentration increases from 0 to 500 mM).

The anionic SDS is a very commonly used and effective surfactant in solubilizing most proteins. It disrupts non-covalent bonds within and between proteins, denaturing them, and resulting in the loss of their native conformation and function. SDS binds to a protein with a ratio of 1.4:1 w/w (corresponding to about one SDS molecule per two amino acids), masking the charge of the protein. Thus SDS adds an overall negative charge to all proteins in the sample regardless of their isoelectric point (pI). Once bound by negatively charged SDS molecules the proteins can be separated based on size. That is a big reason for the wide use of SDS polyacrylamide gel electrophoresis (SDS-PAGE) for separating and studying proteins. Usually, for complete cell lysis in the presence of SDS, a sample must be sonicated or sheared (e.g., passed through a 19G needle) several times to ensure DNA degradation. SDS cannot be used when active proteins are required or when protein-protein interactions are being studied because both of these are disrupted by the SDS. When working with SDS it is important to know that SDS precipitates at low temperatures, and this effect is enhanced in the presence of potassium salts. This phenomenon can sometimes be exploited to remove SDS from a protein sample [4].

Sodium deoxycholate and sodium cholate are bile salts detergents. They are both anionic detergents. These detergents are often used for membrane disruption and membrane protein extraction, for example, apelin receptor [5]. Deoxycholate does denature proteins while cholate is a non-denaturing detergent. One potential benefit to both of these detergents is that they can be removed from samples via dialysis, which may help with quantification and/or downstream analyses of proteins.

Sarkosyl, also known as sarcosyl or sodium lauroyl sarcosinate, is an anionic surfactant. It is amphiphilic due to the hydrophobic 14-carbon chain (lauroyl) and the hydrophilic carboxylate. The carboxylate with a pKa value of 3.6 is negatively charged in any physiological solution. Sarkosyl is prepared from lauroyl chloride and sarcosine in the presence of sodium hydroxide and is purified by recrystallization from alcohol, or by acidification with a mineral acid, separation of the free acid, and neutralization of the free acid. Sarkosyl has also been used to improve wetting and penetration of topical pharmaceutical products. In the food industry, sarkosyl is approved for use in processing, packaging, and transporting food for human consumption, and in adhesives used in food storage or transportation. It is widely used in cosmetic formulations such as shampoos and body washes at concentrations around 3-13% [6]. Sarkosyl is also used in metal finishing end processing for its crystal modifying, anti-rust, and anti-corrosion properties.

Sarkosyl is widely utilized in laboratory experiments, for example for solubilizing tau in Alzheimer disease research [7], due to its good water solubility, high foam stability, and strong sorption capacity to proteins. Sarkosyl serves as a detergent to permeabilize cells and extract proteins in isolation and purification techniques such as western blot and indirect ELISA. It can also inhibit the initiation of DNA transcription.

One major application of sarkosyl is for solubilizing and refolding proteins from inclusion bodies (protein aggregates within cytoplasm or nuclei). Eukaryotic recombinant proteins overexpressed in Escherichia coli tend to form such inclusion bodies. Sarkosyl is often used to solubilize an inclusion body pellet to extract the proteins and allow them to refold into their native form. Earlier work involved solubilizing inclusion bodies with denaturants, such as urea or guanidinium hydrochloride, and refolding by slow dilution — however, most of the solubilized proteins aggregate and precipitate upon removal of the strong detergents. Sarkosyl is an effective solubilizing agent that minimizes aggregation and allows refolding at higher protein concentrations (as much as 10-fold higher when compared to using guanidinium hydrochloride [8] ). One study found the over 95% of inclusion body fusion proteins were solubilized with 10% sarkosyl, and that the proteins could then be recovered with a mix of other detergents (i.e., Triton X-100 and CHAPS) [9]. Proteins in the soluble extract with sarkosyl can also be stored at 4°C for a week before affinity purification. It should be noted, however, that sarkosyl interferes with the subsequent chromatographic process and must be removed from the solution by dilution or dialysis.

Non-ionic detergents have uncharged hydrophilic headgroups. They are considered mild surfactants as they break protein-lipid and lipid-lipid associations, but typically not protein-protein interactions, and generally, do not denature proteins. Therefore, many membrane proteins may be solubilized in their native and active form, retaining their protein interactors. However, because not all proteins behave the same with different non-ionic detergents, trial and error may be necessary to find the best detergent for your protein(s) of interest. Additionally, it should be noted that most non-ionic detergents interfere with ultra-violet (UV) spectrophotometry. Therefore, protein determination at 280 nm in the presence of non-ionic detergents is typically imprecise.

All members of the Triton family: Triton X-100, Triton X-114, Nonidet P-40 (NP-40), Igepal® CA-630, are quite similar, differing slightly in their average number (n) of monomers per micelle (9.6, 8.0, 9.0, and 9.5, respectively) and the size distribution of their polyethylene glycol (PEG)-based headgroup. The CMC values of these detergents are low, and therefore they can not be easily removed by dialysis. Triton X-100, a typical non-ionic detergent, derives from polyoxyethylene and contains an alkylphenyl hydrophobic group. Triton X-100 is commonly used for isolating membrane protein complexes, and the surfactant of choice for most such as for co-immunoprecipitation experiments. Other members of the Triton family are used for membrane protein isolation by phase-separation due to low cloud points (the temperature at which the micelles aggregate and form a distinct phase). While the cloud point of Triton X-100 is 64°C, the cloud point of Triton X-114 is 23°C. This allows for membrane protein extraction and solubilization in Triton X-114 without bringing the samples up to warmer temperatures which may denature many proteins.

Brij™ 35 is another nonionic polyoxyethylene surfactant, commonly used as a component of cell lysis buffers or assay buffers or a surfactant in HPLC applications.

The n-dodecyl-β-D-maltoside (DDM) is a glycosidic surfactant, increasingly used with hydrophobic and membrane protein isolation when the protein activity needs to be preserved. It is more efficient at protein solubilization for 2-D electrophoresis than several other detergents, including CHAPS and NP-40 [10]. The glycochain in its lipophilic site, its high CMC of 0.17 mM and the interface of the micelles create an aqueous-like microenvironment ideal for solubilizing and retaining the stability of membrane and hydrophobic proteins [11]. For example, Winkler MBL et al purified NCR1 protein with the addition of n-dodecyl-β-D-maltopyranoside [12] so did Li Y et al for LptB2FG and LptB2FGC proteins [13]. Steichen JM et al mixed protein complexes in a solution of DDM from Anatrace before Cryo-EM [14].

Other maltosides, such as beta-decyl-maltoside, have different lengths of the hydrophobic alkyl chains. Glucoside (octyl-glucoside) are a potential alternative to maltoside detergents for protein research [15].

Digitonin, a steroidal glycoside derived from the purple foxglove plant (Digitalis purpurea), is used for the solubilization of cellular membranes. As with other non-ionic detergents discussed here, digitonin is frequently used to solubilized membrane proteins without denaturing them. For example, B de Laval et al lysed cells with 1% of digitonin for Tn5 transposase reaction during the ATAC-seq protocol [16]. Zhao Y et al released synaptic and extrasynaptic AMPA receptors from postsynaptic density with digitonin [17]. Additionally, digitonin is used to extract cellular organelles. Digitonin interacts with cholesterol in membranes and thus can be used to permeabilize the cholesterol-rich plasma membrane while leaving the cholesterol-poor organelle membranes intact.

Tween-20 and Tween-80 are polysorbate surfactants with a fatty acid ester moiety and a long polyoxyethylene chain. They have very low CMC, are generally gentle surfactants, do not affect protein activity and are effective in solubilization. Tweens are not common ingredients of cell lysis buffers however, they are routinely used as washing agents in immunoblotting and ELISA to minimize nonspecific binding of antibodies and to remove unbound moieties, and used to permeabilize cell membranes. For example, Yang J et al immunostained intracellular FLAG tag after treating HEK293 cells with 0.2% Tween 20 [18].

One common question regarding the Tween family detergents is the difference between Tween 20 and Tween 80, the two most commonly used members. Tween 20 has lauric acid, while Tween 80 has oleic acid (Figure 3). Table 2 summarizes various aspects between them. These detergents can often be used interchangeably however, the difference between them is sometimes important, such as in in vivo studies that may be influenced by the different levels of hemolytic effect of Tween 20 and Tween 80 [19]. Greenwood DJ et al, for example, grew Mycobacterium tuberculosis in a medium supplemented with 0.05% Tween 80 [20]. Ouadah Y et al injected a dibenzazepine solution with 0.1% v/v Tween 80 into mice to inhibit Notch signalling [21].

Synonyms Chemical Formula Molecular Weight Density (g/mL) Appearance Applications
Tween 20polysorbate 20, polyoxyethylene sorbitan monolaurate, PEG (20) sorbitan monolaurateC 58 H 114 O 26 12281.1Clear, yellow to yellow-green viscous liquida broad range of applications: as a blocking agent in PBS or TBS wash buffers for ELISA, Western blotting and other immunoassay methods for lysing mammalian cells and as a solubilizing agent for membrane proteins.
Tween 80polysorbate 80, polyoxyethylene sorbitan monooleate, PEG (80) sorbitan monooleateC 64 H 124 O 26 13101.06-1.09amber colored viscous liquidas a stabilizing agent for proteins used in tests for the identification of phenotype of some mycobacteria used in vaccine preparations [22]

Though non-ionic detergents are generally relatively mild, many proteins do denature or aggregate in the presence of these detergents. To ameliorate this issue new non-ionic glyco-lithocholate amphiphiles (GLC-1, GLC-2, and GLC-3) and glyco-diosgenin amphiphile (GDN) have been developed [23]. GDN was used to extract yeast mitochondrial dimeric ATP synthase to understand better how the protein functions [24]. Pluronic F-68 is commonly used in suspension cell culture at 0.1% to reduce the water shear force [25].

The headgroups of zwitterionic detergents are hydrophilic and contain both positive and negative charges in equal numbers, resulting in zero net charge. They are more harsh surfactants than the non-ionic detergents. A typical zwitterionic detergent is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, better known as CHAPS. CHAPS high CMC (6 mM at room temperature) allows efficient removal by dialysis. It is very common in sample preparation at concentrations of 2-4% for isoelectric focusing and 2D electrophoresis. CHAPSO differs with CHAPS in that it contains a more polar headgroup, which makes it more capable of solubilizing hydrophobic molecules. Thus, CHAPSO is mainly used for solubilization of integral membrane proteins.

Chaotropic agents are similar substances to surfactants in that they break non-covalent interactions (hydrogen bonds, dipole-dipole interactions, hydrophobic interactions) facilitating protein denaturation, which in this case is usually reversible. Urea is a common chaotropic agent used alone, or in combination with thiourea or other detergents, in applications like 2D-gel electrophoresis and in-solution enzymatic digestion of proteins for preparation during proteomic workflows. When using Urea, extra care must be taken not to heat the sample above 37°C as this will lead to carbamylation of proteins [26].

For membrane protein solubility, a detergent with high CMC should generally be chosen, and the volume and concentration of the buffer are also crucial as enough detergent should be present to solubilize all membrane proteins in the sample. In most cases, the detergent concentration should be well about the CMC level (at least 2X the CMC) to ensure sufficient micelle concentration to solubilize the membrane proteins. According to Linke [3], at least one micelle is needed per membrane protein molecule to sufficiently mimic the lipid environment of a membrane (Fig. 1B, D).

Phase separation can be used to purify the proteins further. This requires adjusting the temperature and the concentrations of salts and detergent in the buffer to cause the detergent micelles to aggregate and separate from the aqueous layer. In this case, the membrane proteins, surrounded by the micelles, aggregate with the detergent. The temperature at which the detergent solution separates into two phases, the cloud point, is affected by glycerol or salts in the buffer (e.g., Triton X-114 has a cloud point of 23°C, but in the presence of 20% glycerol, the cloud point declines to 4°C). This is very important since the stability of a protein is affected by high temperatures.

A good detergent should be able to lyse cells, solubilize proteins and be suitable for your downstream application(s). Also, the solubilized protein in native or denatured form should be considered. There is no ideal detergent for all applications, and even in the same application, the result varies (Table 3). Therefore, after options are considered, trial and error are often necessary to find the best detergent, and a mixture of detergents may be optimal. Also, the fresh preparation of detergent working solution is usually the best practice to avoid hydrolysis and oxidation.

DetergentMW (Da) monomerMW (Da) micelleCMC (mM) 25 o CAggregation No.Cloud Point ( o C)Avg. Micellar WeightStrengthDialyzableApplications
SDS28918,0007-1062>10018,000HarshYesCell lysis, Electrophoresis, WB, hybridization
Triton X-10062590,0000.2-0.9100-1556580,000MildNoEnzyme immunoassays, IP, Membrane solubilization
CHAPS6156,150610>1006,150MildYesIEF, IP
NP-4068090,0000.059 45-50 MildNoIEF
n-dodecyl-β-D-maltoside511 0.1598 50,000 Protein Crystallization
Tween-201228 0.06 76 MildNoWB, ELISA, Enzyme immunoassays
Digitonin122970,000<0.560 70,000MildNoMembrane solubilization

The downstream applications often require that detergent concentrations be lowered or completely removed. For such purposes, size exclusion chromatography or dialysis can be used if the micelle size is substantially different than the protein of interest or micelles are small enough (i.e., high CMC) to pass through the dialysis tubing [3]. Other methods employ the use of detergent binding non-polar beads or resins, cyclodextrin inclusion compounds [27], ion-exchange chromatography or protein precipitation. However, the buffer used after detergent removal must be selected carefully to avoid protein precipitation or aggregation.

Labome surveys the literature for the application of detergents. The following table lists the main suppliers, and the number of articles, indicating most of the detergents are supplied by MilliporeSigma.

detergent suppliers
Triton X-100Thermo Fisher [28, 29], Electron Microscopy Sciences [30], Amresco, JT Baker
Tween-20Bio-Rad [31], MilliporeSigma [29], Thermo Fisher
SDSAmresco, Bio-Rad, Q.BIOgene, MilliporeSigma
NP-40Roche, MilliporeSigma [32]
CHAPSMilliporeSigma, JT Baker
digitoninMilliporeSigma, Wako
DDMGeneron [33], Anatrace [15]

Thermo Fisher Pierce Triton X-100, for exampl, BP151 [29] or 85111 [28], was used to lyse cell and tissue samples for immunohistochesmitry [29] and immunocytochemistry [28]. MilliporeSigma Triton X-100 was used to lyze cells [34], or permeabilize cells in immunocytochemistry [35], and in blocking buffer for immunohistochemistry [36, 37] and proteinase K protection assay [38].

Tween-20 is commonly used in washing buffers, such as TBS-Tween (TBS-T) or PBS-Tween (PBT-T), in various immunoassays. MilliporeSigma Tween-20, for example, P1379 [29], was used in washing blots [29], in IHC experiments (P1379) [39], in immunoprecipitation [40],and in microfluidic array multiplex PCR [41] and others [42]. MilliporeSigma Tween-80, was used to dissolve erlotinib (a chemotherapy drug) [43] and as a supplement to grow M. tuberculosis strains [44].

Lonza SDS (catalog number 51213) was used in chromatin preparations [39]. Amresco SDS was used in SDS-PAGE [45]. Bio-Rad sodium dodecyl sulfate was used to prepare a radioimmunoprecipitation assay buffer [46]. MilliporeSigma-Aldrich SDS was used to prepare buffers for, among others, in vitro octanoylation assays, Laemmli sample buffer, 2D-DIGE experiments [47].

Roche NP-40 was used in cell lysis [48, 49]. MilliporeSigma NP-40 was used to prepare radioimmunoprecipitation assay buffer [46], cell lysis/homogenization buffers buffer [50, 51] and immunoprecipitation assay RIPA buffer [52].

MilliporeSigma CHAPS was used in buffers for protein crystallization [53]. JT Baker CHAPS was used to lyse cells to study viral interaction with human ASF1 protein [54].

MilliporeSigma was used in an immunocytochemistry experiment to study PI4P [55] and used to perform proteinase K protection assays [38], and to extract RNA [56]. Wako digitonin was used to lyse cells [57] and perform immunoprecipitation experiments [58].

Y Lee et al solubilized a GPCR protein with dodecylmaltoside / DDM from Generon [33]. Anatrace n-decyl-beta-D-maltopyranoside was used for protein purification [59, 60] so were its n-dodecyl-beta-D-maltoside [61] and n-undecyl-beta-D-maltoside [62, 63]. Glycon beta-dodecyl-maltoside and beta-decyl-maltoside were also used in protein purification [64]. Anatrace n-octyl-beta-glucoside was used in solubilizing AQP4 proteins [15].

Silva MC et al used Brij-35 as the detergent for bio-layer interferometry biosensor assay [65]. For protein purifications, Affymetrix octyl glucose neopentyl glycol (OGNPG) at 1% [66], and MilliporeSigma cholesteryl hemisuccinate at 0.1% or 0.05% (w/v) [11, 67] were used. For chromatin-related assays, MilliporeSigma-Aldrich sodium deoxycholate (catalog number D6750) and Igepal (catalog number I8896), and TEKnova N-lauroylsarcosine (catalog number S3379) were used [39].


Western blot: technique, theory, and trouble shooting

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Keywords: Bio-medical research protein western blot.

Conflict of interest statement

Conflict of Interest: None declared.

Figures

Assembled rack for gel solidification

Assembled rack for gel solidification

Add gel solution using a transfer pipette

Add running buffer to the…

Add running buffer to the electrophorator

Add samples and molecular marker…

Add samples and molecular marker to the gel, after removing the combs

(a) Samples running through the…

(a) Samples running through the stacking gel (lower voltage). (b): Samples running through…


Contents

Undiluted Triton X-100 is a clear viscous fluid (less viscous than undiluted glycerol). Undiluted Triton X-100 has a viscosity of about 270 centipoise at 25 °C which comes down to about 80 centipoise at 50 °C. Triton X-100 is soluble at 25 °C in water, toluene, xylene, trichloroethylene, ethylene glycol, ethyl ether, ethyl alcohol, isopropyl alcohol, and ethylene dichloride. Triton X-100 is insoluble in kerosene, mineral spirits, and naphtha, unless a coupling agent like oleic acid is used. [5]

Triton X-100 is a commonly used detergent in laboratories. [6] Triton X-100 is widely used to lyse cells to extract protein or organelles, or to permeabilize the membranes of living cells. [7]

Some applications include:

    of lipid-enveloped viruses (e.g. HIV, HBV, HCV) in manufacturing of biopharmaceuticals
  • Industrial purpose (plating of metal) , including Fluzone
  • Permeabilizing unfixed (or lightly fixed) eukaryotic cell membranes [7]
  • Solubilizing membrane proteins in their native state in conjunction with zwitterionic detergents such as CHAPS
  • Part of the lysis buffer (usually in a 5% solution in alkaline lysis buffer) in DNA extraction
  • Reducing surface tension of aqueous solutions during immunostaining (usually at a concentration of 0.1-0.5% in TBS or PBS Buffer)
  • Dispersion of carbon materials for soft composite materials
  • Restricting colony expansion in Aspergillus nidulans in microbiology
  • Decellularization of animal-derived tissues
  • Removing SDS from SDS-PAGE gels prior to renaturing the proteins within the gel
  • Disruption of cell monolayers as a positive control for TEER measurements
  • Micellar catalyst

Apart from laboratory use, Triton X-100 can be found in several types of cleaning compounds, [8] ranging from heavy-duty industrial products to gentle detergents. It is also a popular ingredient in homemade vinyl record cleaning fluids together with distilled water and isopropyl alcohol. [9]

In December 2012, the European Chemicals Agency (ECHA) included the substance group “4-(1,1,3,3-tetramethylbutyl)phenol, ethoxylated” – which includes Triton X-100 – in the Candidate List of substances of very high concern [10] of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation which addresses the production, import and use of chemical substances and their potential impacts on human health and the environment. [11] A Triton X-100 degradation product has indeed turned out to be ecotoxic as it possesses hormone-like (estrogeno-mimetic) activity that may act on wildlife. [12] The ECHA finally included the substance group in the Authorisation List (Annex XIV), [13] mandating the pharmaceutical and other industries to replace this detergent by the “sunset date” January 4, 2021, thereby affecting EU manufacturers, importers, and downstream users, as well as non-European manufacturers exporting their products into the EU.

Alternatives for viral inactivation Edit

Since the inclusion of Triton X-100 in the candidate list of substances of very high concern for authorization, pharmaceutical companies, as well as bioprocessing research groups, are in need of an alternative detergent which must at the same time be eco-friendly and effective. Ideally, a Triton X-100 replacement should generate minimal manufacturing process change, because only then the necessary updates of regulatory filings for medicines could be realized without additional animal experiments or even clinical studies. Therefore, an alternative virus-inactivating detergent should have physico-chemical properties similar to Triton X-100, should be soluble, easy to remove, eco-friendly, but not degrade to toxic metabolites. In a recent study, [14] two alternatives for antiviral treatment in biopharmaceutical manufacturing have been identified: Triton X-100 reduced, as well as a novel compound which was named Nereid (after the mermaids in Greek mythology). As reflected by the name, Nereid can be seen as just another relative of the Triton X-100 family, however, due to a small molecular difference, it does not degrade into phenolic compounds the way that Triton X-100 does. The virus inactivation studies comprised experiments with several relevant viruses under various conditions. It turned out that at room temperature, where most virus inactivation steps in biopharmaceutical manufacturing are conducted, both Triton X-100 reduced and Nereid showed similar virus inactivating performances as Triton X-100. In contrast, for some processes that are conducted at cold temperatures, Nereid and Triton X-100 gave better results than Triton X-100 reduced. To date, Nereid can be produced at kilogram-scale using a three-step synthesis, and a patent has been applied for. [15] Nereid is scalable and compatible with existing processes and has not shown any impact on product activity so far. In terms of performance, Nereid would be a robust “all-in-one” replacement for Triton X-100. Thus, it is currently tested in ecotoxicology and biodegradation studies to confirm that it is environmentally safe.


Chapter 13 - Co-IP assays for measuring GPCR–arrestin interactions

βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR–arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR–arrestin interaction in cells.


Interpretation and Use of the Western Blot Assay for Serodiagnosis of Human Immunodeficiency Virus Type 1 Infections

Reported by: Association of State and Territorial Public Health Laboratory Directors and AIDS Program, Center for Infectious Diseases, Public Health Practice Program Office, Centers for Disease Control* The Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and CDC have collaborated in preparing this report. It includes a description of various interpretive criteria associated with the Western blot test for HIV-1, evaluates the sensitivity and specificity of these criteria as tools for public health practice, and provides recommendations for use of the Western blot and the manner in which to report results in order to provide clinicians and public health policy officials with useful information in their efforts to reach an accurate diagnosis for persons tested for HIV-1 infection. INTRODUCTION

The development of sensitive and specific tests for antibody to human immunodeficiency virus type 1 (HIV-1) progressed rapidly after this retrovirus was identified as the cause of acquired immunodeficiency syndrome (AIDS). These tests have been used for various purposes, including clinical diagnosis of HIV-1 infection--for symptomatic and asymptomatic patients in counseling and testing programs--for seroprevalence surveys, and for blood-donor screening.

Enzyme immunoassay (EIA) is the most widely used serologic test for detecting antibody to HIV-1. Serum samples that are repeatedly reactive in the EIA for HIV-1 antibody are then retested with a supplemental and more specific test, the most common of which is the Western blot (1-3). To date, only one commercial Western blot test (Du PontœPr) has been licensed by the Food and Drug Administration (FDA). The purpose of this report is to provide guidance for interpreting Western blot test results and their use in diagnosing HIV-1 infection. THE WESTERN BLOT ASSAY

The Western blot assay is a method in which individual proteins of an HIV-1 lysate are separated according to size by polyacrylamide gel electrophoresis. The viral proteins are then transferred onto nitrocellulose paper and reacted with the patient's serum. Any HIV antibody from the patient's serum is detected by an antihuman immunoglobulin G (IgG) antibody conjugated with an enzyme that in the presence of substrate will produce a colored band. Positive and negative control serum specimens are run simultaneously to allow identification of viral proteins.

Table 1 lists the major structural proteins coded for by the HIV genome. Antibodies to the HIV-1 major group-specific antigen (GAG) protein p24, and its precursor p55, are the earliest detected after infection by Western blot and tend to decrease or become undetectable with onset or progression of clinical symptoms (4-9). In contrast, antibodies to the envelope (ENV) precursor protein gp160 and the final ENV proteins (gp120 and gp41) can be detected in specimens from virtually all HIV-infected persons regardless of clinical stage (4-9). Antibodies to the polymerase (POL) gene products (p31, p51, and p66) are also commonly detected if these antigens are present on the Western blot strips. However, in a recent study, the protein with a mobility of 160 kilodaltons (kd) present in commercially available Western blots and in viral lysate antigen preparations was identified as a multimer of the gp41 protein (10,11). Furthermore, this study presented evidence that the reaction observed against the gp120 on certain Western blots may have resulted in part from a reaction with a multimeric form of the gp41. In fact, the true gp120 was shown to be absent from some commercial Western blot antigens. When these reagents were used, serum specimens with only gp41 antibodies produced bands at the 41-, 120-, and 160-kd positions. Interpretive Criteria

Although the overall sensitivity and specificity of the Western blot for detection of antibodies to the various viral proteins are high, there has been substantial debate regarding the interpretive criteria. The currently licensed Du Pont Western blot test specifies that the test result should be interpreted as positive only when the detected bands include p24 and p31, and gp41 or gp120/160 (12) (see Table 2). Conversely, a negative Du Pont Western blot test result requires the absence of any and all bands--not just viral-bands. All other patterns are regarded as indeterminate. This interpretation scheme maximizes the specificity of the assay and is mainly intended for use with samples from persons, such as blood donors, for whom there is usually little clinical or virologic information available. (Donated units of blood that are repeatedly reactive by EIA are discarded Western blot results are used to guide donor notification and deferral.) These criteria are not ideal for all situations, especially the testing of persons at increased risk for HIV infection, or with symptoms suggestive of this infection.

Alternative criteria have been proposed by various groups. ASTPHLD has proposed that a positive test result be defined by the presence of any two of the following bands: p24, gp41, and gp120/160 (13). The Consortium for Retrovirus Serology Standardization (CRSS) has defined a positive test result as the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160) (14). The American Red Cross has defined a positive test result as greater than or equal to 1 band from each of the GAG, POL, and ENV gene-product groups (15). These three groups and DuPont all agree that an indeterminate result is the presence of any other band or bands that fail to meet the positive criteria, and that a negative result is the absence of all bands.

The criteria for a negative Western blot interpretation specify "no bands." This interpretation is essential because some observed bands may reflect the presence of antibodies to HIV regulatory proteins or may indicate partially processed or degraded viral structural proteins. Furthermore, different Western blots (commercial, as well as "in-house" preparations) and different virus-antigen preparations used to prepare Western blots may contain different numbers and concentrations of both viral-specific and contaminating cellular proteins that may have unpredictable molecular weights. Evaluation of Criteria

To compare the four sets of criteria for Western blot interpretation, CDC selected 424 serum samples on the basis of the patients' clinical status and EIA results only, and analyzed them using the licensed Du Pont Western blot test (CDC unpublished data). The samples were scored according to each of the criteria (Table 3). For all three categories with repeatedly reactive EIA test results, the Western blot results demonstrate that the ASTPHLD definition gives the highest percentage of positive and the lowest percentage of indeterminate results. The interpretive standards that require the identification of bands from each of the three groups of gene products tend to have indeterminate results for some AIDS and other symptomatic patients due to absence of antibodies to p24 (n=5) or to p31 (n=14) or absence of both types of antibodies (n=2). Since these patients clearly are infected with HIV, the three-gene-product approach to Western blot interpretation is not sensitive enough for public health or clinical practice.

The ASTPHLD/CDC criteria for a positive Western blot differ from the CRSS criteria in two ways: first, ASTPHLD/CDC deletes p31, a change that does not affect the sensitivity or specificity of the criteria (Table 3), and second, ASTPHLD/CDC adds "gp41 and gp120/160," a combination not interpreted as positive with the CRSS criteria. This latter combination of bands represents antibody to envelope glycoproteins only. In practice, this is a rare finding for asymptomatically infected persons, but it has been reported to be specific for HIV-infected persons and should be included in the positive criteria (9). However, when a Western blot test has only the multimeric form of gp41 and no true gp120 present, a serum sample would be scored as positive on the basis of the presence of antibody to a single envelope glycoprotein, gp41. HIV-1-infected persons with this profile have lost their antibodies to the GAG proteins and are usually symptomatic and do not present a diagnostic problem.

The ASTPHLD/CDC interpretive criteria for a negative result are identical to the FDA recommendation for blood-donor reentry or the Western blot interpretive criteria that are specified in the licensed Western blot kit package insert. RECOMMENDATIONS

On the basis of the results described above, CDC concurred with the ASTPHLD criteria and recommends their use in public health and clinical practice.

Laboratories should report test results as positive, indeterminate, or negative. The Public Health Service recommends that no positive test results be given to clients/patients until a screening test has been repeatedly reactive (i.e., greater than or equal to two tests) on the same specimen and a supplemental, more specific test such as the Western blot has been used to validate those results (3). Upon request, laboratory reports may also contain a list of the bands detected and reference to the interpretive criteria the laboratory uses. Because of the variability of unlicensed reagents, laboratories using non-FDA-licensed Western blots should compare, on a routine basis, their tests with the FDA-licensed Western blot kit using well-characterized serum specimens.

Clinical diagnosis and follow-up of patients is the responsibility of the clinical practitioner. Serologic test results are but one contribution to a patient's data base, which contains medical history (including high-risk behavior or exposure to HIV), results of physical examination, and other clinical findings. Clinicians must consider the total profile for a client when attempting to make a diagnosis after indeterminate Western blot results have been obtained. Accurate diagnosis for such persons can be challenging--and the challenge can be complicated by the tendency of some clients to become distressed by the apparent "uncertainty" of their test results.

Clinical follow-up of patients with indeterminate Western blot results may require many months of observation, interviewing, and testing. Most indeterminate patterns involve p18 (also referred to as p17), p24, or p55, or any combination of these three proteins (16-18). In one study of 390 "atypical" or indeterminate samples, 53% reacted against p24, with or without p18 or p55 47% reacted against p18 (but not p24), with or without p55 (18).

Some indeterminate results may be obtained with serum samples from persons who are in the process of seroconverting. A compilation of 209 volunteer blood donors with GAG-only indeterminate Western blot results were followed for as long as 2 years (17-21). During that time, only five of 134 persons who had initially reacted to p24 developed additional bands on the Western blot test. None of the 75 persons who initially reacted against p18 (but not p24) developed additional bands. The five persons who did seroconvert had positive results when their first follow-up samples were tested. The intervals between initial and follow-up tests were 8 weeks (two persons), 20 weeks (two persons), and 32 weeks (one person). The three longest intervals reflected delays in follow-up testing and not the actual time to seroconversion. These results do not refute earlier findings that seroconversion typically occurs within 3 months of infection (5,22). The importance of careful risk assessment for persons with indeterminate Western blot patterns was reemphasized when in one study (18) two of three people who initially had indeterminate results (but later seroconverted) disclosed histories of risk behavior when they were reinterviewed during follow-up.

A person whose Western blot test results continue to be consistently indeterminate for at least 6 months--in the absence of any known risk factors, clinical symptoms, or other findings--may be considered to be negative for antibodies to HIV-1. Such persons should be reassured that they are almost certainly not infected with HIV-1. However, no large-scale studies have been done to provide virologic data to confirm independently the serologic findings from the studies of clients whose Western blot test results are consistently indeterminate. In contrast, an asymptomatic person who has an indeterminate Western blot test result and a history of possible exposure to or symptoms compatible with HIV infection requires additional diagnostic follow-up. This should include conducting serial Western blot testing, assessing the function of the individual's immune system, and eliciting the cooperation of the person's sexual and needle-sharing partners to determine whether they are infected. Individuals with a pattern of indeterminate Western blot test results should not donate blood or plasma for either transfusion or use in manufactured blood products.

As the HIV/AIDS epidemic continues, additional tests of higher specificity will be needed to decrease the number of false-positive reactions and to permit correct diagnosis of HIV infection in a larger spectrum of clinical situations in which an indeterminate antibody profile exists. The use of new antibody tests based on antigens derived by recombinant deoxyribonucleic acid (DNA) technology or the application of DNA probe technology--particularly DNA amplification by the polymerase chain reaction (PCR)--already shows promise in this area (23).

References

Centers for Disease Control. Provisional public health service

inter-agency recommendations for screening donated blood and plasma for antibody to the virus causing acquired immunodeficiency syndrome. MMWR 198534:1-5.

2. Centers for Disease Control. Public health service guidelines for counseling and antibody testing to prevent HIV infection and AIDS. MMWR 198736:509-15.

3. Centers for Disease Control. Update: Serologic testing for antibody to human immunodeficiency virus. MMWR 198836:833-40, 845.

4. Lange JMA, Coutinho RA, Krone WJA, et al. Distinct IgG recognition patterns during progression of subclinical and clinical infection with lymphadenopathy associated virus/human T lymphotropic virus. Brit Med J 1986292:228-30.

5. Esteban JI, Shih JW, Tai CC, et al. Importance of Western blot analysis in predicting infectivity of anti-HTLV-III/LAV positive blood. Lancet 19852:1083-6.

6. Goudsmit J, Lange JMA, Paul DA, et al. 1987. Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J Infect Dis 1987155:558-60.

7. Lange JDA, Paul DA, Huisman HG, et al. Persistent HIV antigenemia and decline of HIV core antibodies associated with transition to AIDS. Brit Med J 1986293:1459-62.

8. Weber JN, Clapham PR, Weiss RA, et al. Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis. Lancet 1987i:119-21.


Maintain some integrity with NP-40 or Triton X-100 lysis buffer

NP-40 (Nonidet P-40) and Triton X-100 are milder, nonionic detergents. They are good at solubilizing membrane proteins and for isolating cytoplasmic proteins. Proteins retain their native state in the presence of these detergents and protein-protein interactions can be preserved. These buffers can be used for co-IPs.

NP-40 and Triton X-100 will not lyse nuclear membranes. After lysis, pellet the nuclei by centrifugation and transfer the supernatant to a new tube. If you wish to isolate both the nuclear and soluble fractions, resuspend the nuclear pellet in RIPA buffer.

NP-40 is also marketed under the name Igepal CA-630.

NP-40/Triton X-100 lysis buffer : 50 mM Tris•HCl, pH 8.5, 150 mM NaCl*, 1% detergent*

*The concentrations of salts and detergents can be altered to optimize protein recovery.


Primary antibodies and determining specificity

The principle of the WB is the detection of protein(s) through the binding and recognition of antibodies (Ab) to one or more targets this interaction should be highly specific between a portion of the antigen (protein) or epitope and the specific recognition sites found on the fragment antigen-binding (Fab) region of the antibody termed a paratope (Kurien et al., 2011 ) (Fig. 1). The 1°Ab should be thoroughly assessed and validated to be specific and sensitive enough to detect the intended target protein. It is important to check that the antibody is specific toward the native or denatured protein, as the denaturing treatment of protein samples prior to SDS-PAGE may alter the exposure and availability of the epitope, affecting antibody binding affinity. In some cases, it may be necessary to use “native-specific” monoclonal antibodies (Tino et al., 2000 ). The targeted peptide sequence may be available from the supplier to allow confirmation of specificity and region of binding however, occasionally this may be unavailable proprietary information. Traditionally 1°Ab are produced through immunization of the host using purified target proteins, whereas modern approaches utilize synthetic peptides, often producing Ab toward short denatured 8–10 amino acid sequences. Isolation and purification is generally achieved through affinity chromatography isolating antibodies and small proteins and/or anion-exchange filtration depending on the class (i.e., IgG, IgM) (Clezardin et al., 1986 ). Predicted and confirmed species cross-reactivity information is often only available through the vendor however, binding will entirely depend on the antigen region. When choosing a 1°Ab, there may be multiple forms available from different vendors, ideally each would bind to a unique antigen upon the protein of interest, allowing accurate assessment of the protein's abundance. However, this is often not the case, and assessment of the previous literature utilizing that antibody is strongly advised. Some proteins may be orthologous and contain the same or similar sequence to other species. Thus, if a 1°Ab is specific to an epitope with this sequence, it may be used to probe other species (i.e., GAPDH 1°Ab may be used on human, rat, and mouse tissue). Depending on the protein of interest, extensive testing of multiple antibodies may have already been undertaken, allowing the most suitable antibody to be selected. For example, the VDR has low expression within skeletal muscle, and in order to find a 1°Ab capable of detecting it by WB and immunofluorescence, extensive validation of a panel of multiple 1°Abs was required (Wang et al., 2010 ). The identification of a highly specific VDR 1°Ab (D-6 Santa Cruz Biotechnology, Cambridge, UK) was confirmed and is believed to be the most representative. Assessment of new antibodies for a target antigen requires even more careful testing, including the use of a variety of appropriate positive and negative controls (discussed below).

Specificity and performance of the 1°Ab antibody is also dependent on whether it is monoclonal (mAb) or polyclonal (pAb). Both have disadvantage/advantage pAb are produced from differing B-cell lineages, recognizing multiple epitope regions on an antigen. They are generally more cost-effective (Lipman et al., 2005 ) and provide more antibody molecules that can target the protein of interest, producing potentially a greater level of sensitivity upon analysis (MacPhee, 2010 ). However, their specificity can also be compromised, due to greater possibility of non-specific binding (MacPhee, 2010 ). In contrast, mAb provide highly consistent and specific binding to a specific and known epitope on an antigen, as they are produced from a single cell lineage, raised against a single specific epitope (Lipman et al., 2005 MacPhee, 2010 ). Yet binding affinities of mAb can suffer if the epitope structure is affected in any way through denaturing or electrophoresis for example (Lipman et al., 2005 ).

Depending on the primary amino acid sequence of the target protein, similar epitopes may be present within degradation products or alternate isoforms, potentially presenting additional bands. Degradation products will migrate ahead of the band of interest, due to the decreased molecular weight. 1°Ab affinities toward alternative isoforms may not interfere with data interpretation if the bands are sufficiently separated (i.e., have different molecular weights). For example, certain antibodies toward P70 S6K1 (70 kDa), a critical protein in the mRNA translational initiation pathway and one of the most probed of all in the muscle and exercise field, may bind to the isoform P80 S6K (80 kDa). P80 S6K encodes a nuclear localization signal and contains an additional 23 amino acids and would be present above P70 S6K1 when blotted (Thomas, 1993 ). Nonetheless, sufficient electrophoretic separation between the two isoforms and appropriate controls may allow correct identification. For example, insulin-treated L6 myotubes increased P70 S6K1 phosphorylation (Somwar et al., 1998 ) but not P85 S6K allowing, in this instance, identification of the correct band nonetheless, this does not guarantee specificity. However, the assessment of a bands molecular weight may not always be suitable, as some proteins may migrate to a non-predictable region. For instance, the mTOR regulator REDD1 has a predicted molecular weight of 25 kDa however, it is detectable at 35 kDa due to multiple lysine residues (increased positively charged residues) (Chang et al., 2009 ). This highlights the importance of knowing the migrating properties of the target protein, and if unexpected bands occur, literature investigation may be required.

In order to validate the specificity of a new 1°Ab, it should be tested against a positive lysate or purified protein control, giving a detectable band at the correct molecular weight and a negative sample from a tissue known not to express the intended target [The Human Atlas provides reliable protein expression data (http://www.proteinatlas.org)], resulting in no detectable band. Sometimes, it may be appropriate to include a specific knock-in/out (e.g., via shRNA or siRNA) sample to allow confirmation of a target within the same tissue type. For example, overexpression of AKT isoforms (an essential signaling protein for muscle hypertrophy/atrophy) within rat skeletal muscle following shRNA produced detectable bands at the predicted molecular weight (

40 kDa) compared with control samples (Cleasby et al., 2007 ). Conversely, knockdown of AKT, again within skeletal muscle, demonstrated a reduction in band intensity at the same molecular weight compared with control samples. Similarly protein inhibitors (e.g., LY294002, rapamycin, etc.) known to block specific phosphorylation pathways can be used. For example, the addition of LY294002 to cultured cells inhibits PI(3)K, resulting in decreased phosphorylation of down-stream intermediates (i.e., AKT/P70 S6K1) (Rommel et al., 2001 ). Thus, if probing for phosphorylated P70 S6K1, cultured L6 cells treated with/without insulin and LY294002 will provide a robust positive (greater band intensity) and negative control (reduced intensity), respectively, compared with untreated samples. In this instance, the inclusion of an untreated sample alongside an inhibited negative control will confirm the reduction in band intensity is in fact due to decreased expression, rather than a loss of detection. Despite rigorous testing of antibodies with appropriate positive/negative controls, additional bands may still be present or insufficiently separated making identification and quantitation of the correct band (if present) unreliable. Absolute confirmation of the presence of a protein in a given band may be achieved by mass spectrometry via determination of the peptide sequence (Trauger et al., 2002 ). Briefly, the band of interest is excised and the mixture of proteins digested by trypsin into small peptide sequences capable of being sequenced by liquid chromatography–mass spectrometry (LC-MS/MS). Utilizing this approach, however, requires access to highly specific and costly equipment and technical expertise, but can provide validation of antibody specificity. Ultimately, positive and negative controls will help establish the degree of non-specific binding and potential false-positive bands, along with the confirmation of increased/decreased protein expression, giving confidence that the highlighted band is indeed the correct one.


Western blot protocol

Reviewed December 14 2020

Western blotting is a technique that uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and the application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest and visualized using secondary antibodies and detection reagents.

Contents

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​Solutions and reagents: lysis buffers

These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.

NP-40 buffer

  • 150 mM NaCl
  • 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
  • 50 mM Tris-HCl, pH 8.0
  • Protease inhibitors

RIPA buffer (radioimmunoprecipitation assay buffer)

  • 150 mM NaCl
  • 1% IGEPAL CA-630
  • 0.5% sodium deoxycholate
  • 0.1% SDS (sodium dodecyl sulphate)
  • 50 mM Tris-HCl, pH 8.0
  • Protease inhibitors

Tris-HCl

​ Solutions and reagents: running, transfer, and blocking buffers

​Laemmli 2X buffer/loading buffer

  • 4% SDS
  • 10% 2-mercaptoethanol
  • 20% glycerol
  • 0.004% bromophenol blue
  • 0.125 M Tris-HCl

Check the pH and adjust to 6.8

Running buffer (Tris-Glycine/SDS)

Check the pH and adjust to 8.3

Transfer buffer (wet)

  • 25 mM Tris base
  • 190 mM glycine
  • 20% methanol
  • Check the pH and adjust to 8.3

For proteins larger than 80 kDa, we recommend that SDS is included at a final concentration of 0.1%.

Transfer buffer (semi-dry)

  • 48 mM Tris
  • 39 mM glycine
  • 20% methanol
  • 0.04% SDS

Blocking buffer

3–5% milk or BSA (bovine serum albumin)

Add to TBST buffer. Mix well and filter. Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.

​Sample lysis

​Preparation of lysate from cell culture

  1. Place the cell culture dish on ice and wash the cells with ice-cold PBS.
  2. Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 10 7 cells/100 mm dish/150 cm 2 flask 0.5 mL per 5x10 6 cells/60 mm dish/75 cm 2 flask).
  3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Alternatively, cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in a microcentrifuge tube.
  4. Maintain constant agitation for 30 min at 4°C.
  5. Centrifuge in a microcentrifuge at 4°C. You may have to vary the centrifugation force and time depending on the cell type a guideline is 20 min at 12,000 rpm but this must be determined for your experiment (leukocytes need very light centrifugation).
  6. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.

​Preparation of lysate from tissues

  1. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
  2. Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80°C for later use or keep on ice for immediate homogenization. For a

Sample preparation

  1. Remove a small volume of lysate to perform a protein quantification assay. Determine the protein concentration for each cell lysate.
  2. Determine how much protein to load and add an equal volume 2X Laemmli sample buffer.​

​Loading and running the gel

  1. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with a molecular weight marker. Load 20–30 μg of total protein from cell lysate or tissue homogenate, or 10–100 ng of purified protein.
  2. Run the gel for 1–2 h at 100 V.

The time and voltage may require optimization. We recommend following the manufacturer’s instructions. A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet.

The gel percentage required is dependent on the size of your protein of interest:

Protein size

Gel percentage

Gradient gels can also be used.

​Transferring the protein from the gel to the membrane

The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. The time and voltage of transfer may require some optimization. We recommend following the manufacturer’s instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.

Prepare the stack as follows:

Figure 1. Example of prepared stack.

Antibody staining

  1. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking buffer.
  2. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer. We recommend overnight incubation at 4°C other conditions can be optimized.
  3. Wash the membrane in three washes of TBST, 5 min each.
  4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
  5. Wash the membrane in three washes of TBST, 5 min each.
  6. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
  7. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

Useful links

All lanes: beta Actin antibody - loading control (ab8227) at 1/5000 dilution

Lane 1: HeLa whole cell extract
Lane 2: Yeast cell extract
Lane 3: Mouse brain tissue lysate

Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products your results from using protocols outside of these conditions may vary.

Webinar transcript​

The purpose of western blotting is to separate proteins on a gel according to the molecular weight. The proteins are then transferred onto a membrane where they can be detected using antibodies. Heat the samples and 95 degrees C for five to 10 minutes in a sample buffer containing a reducing agent such as beta-mercaptoethanol. This results in linearized proteins with a negative charge proportional to their size.

Place a gel into the electrophoresis tank and add in buffer, ensuring the tops of the wells are covered. Acrylamide percentage of the gel being used depends on the molecular weight of the target protein. Node a molecular weight market into the first lane then load the samples into adjacent wells. All the samples which contained equal amounts of protein. Once all the samples are loaded, ad running buffer, place the lid onto the electrophoresis tank. Turn on the power supply and set the voltage recommended by the manufacturer of the gels in the gel tank. You should be able to see bubbles rising through the tank. Run the gel until the die front has moved sufficiently down the gel.

The next stage is to transfer the proteins from the gel onto a membrane. Membranes are usually made from nitrocellulose or PVDF. Remove the gel from the tank and carefully release it from its plastic case. Cut up the wells and the gel foot and place the gel into transfer buffer. Prepare the transfer stack by sandwiching the membrane and gel between filter paper and sponges. The membrane should be traced to the positive electrode and the gel closest to the negative electrode. Use a small roller to remove any bubbles between the gel and the membrane. Cap the transfer case closed and submerge into a transfer tank containing transfer buffer. Add water to the outer chamber to keep the system cool and put on the lid. Turn on the power supply to begin protein transfer. Time and voltage require optimization, so check the manufacturer's instructions for guidance.

Now that the proteins have migrated from the gel onto the nitro cellulose membrane, the protein of interest can be detected as an antibody. The membrane can be removed from the cassette and the molecular weight market should now be visible. If required, the transfer of proteins can be confirmed by staining the membrane with [inaudible 00:04:40] solution. To prevent nonspecific binding of the antibody, the membrane needs to be blocked. Pour blocking buffer onto the membrane and agitate gently on a rocker. Typically, this is done using a solution of five percent milk or bovine serum albumin, BSA, for two hours at room temperature or overnight at four degrees. The time and type of blocking buffer should be optimized, so check the data sheet of the primary antibody you intend to use for details.

After the membrane is blocked, remove the blocking buffer and add the diluted primary antibody in the same solution. Incubate on the rocker as before. Typically primary antibody incubations are for one hour at room temperature or overnight at four degrees C. Antibody concentration and incubation time will need to be optimized. Refer to the antibody datasheet for guidance. Pour off the primary antibody and rinse the membrane twice in wash buffer. Follow with one 15 minute wash and three 10 minute washes on a rocker. The wash buffer is usually Trys buffered saline, TBS, or phosphate buffered, saline, PBS, with 0.1 percent tween 20.

Pour off the wash buffer and incubate the membrane in conjugating secondary antibody which has been diluted in blocking buffer. Usually this is done for one hour at room temperature, but antibody concentration and incubation time will need to be optimized. Pull off the secondary antibody and wash the membrane has shown previously.

There are several different systems for detection. If the secondary antibodies conjugate into an enzyme, incubate the membrane in the appropriate substrate before imaging. If the secondary antibodies are fluorescent counjugates then you can move directly onto the imaging step. Imaging can be carried out with x Ray film or with a digital imaging system. Place the membrane into an imaging tray. Place the imaging tray into imaging system. Exposure times will most likely need to be optimized in order to clearly detect the bands relating to the proteins of interest.


Watch the video: Daughters of Triton - The Little Mermaid (August 2022).