Western Blotting

By Rachel Stewart, PhD

Table of Contents

Overview of Western Blotting

History

It may be surprising to learn that the history of the western blot only stretches back to the late 1970s. In fact, essential western blot methodology, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [1] and monoclonal antibodies with antigen specificity [2], were only established in 1970 and 1975, respectively. In 1975, Edwin Southern invented the eponymously-named “southern blot”, a technique in which DNA fragments are separated through electrophoresis based on their size and then transferred to a nitrocellulose membrane for detection [3]. This method was quickly followed two years later by the invention of the “northern blot”, which could detect specific RNA molecules using radio-labeled DNA probes [4].

Inspired by the northern blot, W. Neal Burnette, a postdoc at the Fred Hutchinson Cancer Research Center, developed a method for visualizing proteins separated by SDS-PAGE using monoclonal antibodies. He subsequently termed this method the “western blot”, in a nod to its predecessors. Interestingly, the manuscript detailing this technique was initially rejected, with reviewers criticizing the method’s name as “flippant and frivolous whimsy” [5]. Despite this, the paper was widely circulated and eventually published in 1981 [5]. The research groups of George Stark at Stanford University and Harry Towbin at Friedrich Miescher Institut in Switzerland published similar immunoblotting techniques at roughly the same time [6,7]. The western blot, also commonly known as immunoblot, has since become an essential and ubiquitous technique in biology and medical labs around the world.

Theory and Practical Considerations

Western blotting relies on the electrophoretic separation of proteins from a complex mixture based on their mass, the transfer of these proteins to a solid matrix, and the detection of specific proteins of interest on the matrix using antibodies.

Figure 1: The flow-through of a typical western blot experiment.

In this example, a lysate sample is prepared from cultured cells. The sample is then subject to gel electrophoresis; allowing the proteins in the mixture to be separated according to their size. The proteins are then transferred to a solid-phase membrane. A specific protein of interest is then detected on the membrane using antibodies that recognize an epitope in the target protein, horseradish peroxidase (HRP)-conjugated secondary antibodies, and a chemiluminescent-based detection substrate.

Overview of Western Blotting - Antibodies.com

Sample Preparation

Sample Lysis

A number of protein-containing samples can be applied to immunoblotting, including: whole cell extracts, tissue extracts, and subcellular fractions. Tissue samples are more difficult to prepare, as they must be homogenized or sonicated to ensure cell lysis, and additional specialized preparation techniques may be required. In all cases, the cells must be lysed and the proteins solubilized. Once lysis begins, enzymes that can induce proteolysis and dephosphorylation are released, as such, lysis is typically performed on ice and in the presence of protease inhibitors (Table 1), to prevent protein degradation, and phosphatase inhibitors (Table 2), to prevent dephosphorylation.

Table 1: Common protease inhibitors included in lysis buffer.

Protease Inhibitor Targets Stock
Aprotinin, 2 µg/ml Trypsin, Chymotrypsin, Plasmin. Dilute in water to 10 mg/ml; freeze aliquots.
EDTA, 1-10 µg/ml Mg2+ and Mn2+ metalloproteases. Dilute in deionized water to 0.5M.
EGTA, 1 mM Ca2+ metalloproteases. Dilute in deionized water to 0.5M.
Leupeptin, 1-10 µg/ml Lysosomal proteases. Dilute in water; freeze aliquots.
Pepstatin A, 1 µg/ml Aspartic proteases. Dilute in methanol to 1 mM; freeze aliquots.
PMSF, 1 mM Serine proteases. Dilute in isopropanol; freeze aliquots.

Table 2: Common phosphatase inhibitors included in lysis buffer.

Phosphatase Inhibitor Targets Stock
β-glycerophosphate, 1-2 mM Serine and threonine phosphatases. Dilute in water; freeze aliquots.
Sodium orthovanadate, 1 mM Tyrosine phosphatases. Dilute in water; prepare in a fume hood.
Sodium pyrophosphate, 1-2 mM Serine and threonine phosphatases. Dilute in water; freeze aliquots.
Sodium fluoride, 5-10 mM Serine and threonine phosphatases. Dilute in water; freeze aliquots.

Choosing a Lysis Buffer

The formulation of the lysis buffer will depend on the subcellular location of the protein of interest, as well as on whether the antibody’s epitope is still present on the protein once it has been denatured. In general, denaturing conditions are used for cell lysis, however, if the antibody only recognizes the target protein in its native state, do not use SDS in the electrophoresis procedure and do not heat the sample. The epitope information and / or suggested immunoblotting conditions should be indicated on the antibody’s data sheet.

Distinct subcellular fractions of a biological sample can be isolated by varying the strength of the detergent in the lysis buffer (Table 3). NP-40 buffer or Triton X-100-containing buffer can be used to prepare whole cell extracts, cytoplasmic extracts, and membrane-bound extracts, however, hydrophobic proteins may remain insoluble under these conditions. Zwitterionic detergents, such as CHAPS, are better able to solubilize membrane proteins. RIPA (radioimmunoprecipitation assay) buffer, which contains SDS, is well suited for preparing whole cell extracts, membrane-bound extracts, and nuclear extracts, but disrupts protein-protein interactions.

In some cases, chaotropic agents that disrupt hydrogen bonding, such as 8M urea, can be used to isolate difficult to solubilize proteins [8], however, these samples should not be heated as urea can be hydrolyzed to cyanate, which can chemically modify the protein sample.

Table 3: Select an appropriate lysis buffer based on the target protein’s localization.

Subcellular Fraction Buffer
Whole cell NP-40 or RIPA
Membrane-bound proteins RIPA
Cytoplasmic Tris-HCl
Nuclear RIPA or perform nuclear fractionation.
Mitochondria RIPA or perform mitochondrial fractionation.
Cytoskeleton Tris-Triton

If native lysis conditions are required, proteins may be solubilized by varying the pH and ionic strength of the lysis buffer. Many proteins are soluble at a higher pH. High salt concentrations can also be used to promote protein solubility, although excess salt can produce wavy bands during electrophoresis. If a detergent-free lysis buffer is used, the sample should be homogenized using a Dounce homogenizer, sonicated, or passed through a 28-gauge needle. Lysates containing nuclear extracts should also be passed through a needle, sonicated, or treated with endonuclease to break down DNA and carbohydrates and reduce sample viscosity. Alternatively, protein samples may be purified and concentrated using TCA/acetone protein precipitation or buffer exchange.

If native lysis conditions are required, proteins may be solubilized by varying the pH and ionic strength of the lysis buffer. Many proteins are soluble at a higher pH. High salt concentrations can also be used to promote protein solubility, although excess salt can produce wavy bands during electrophoresis. If a detergent-free lysis buffer is used, the sample should be homogenized using a Dounce homogenizer, sonicated, or passed through a 28-gauge needle. Lysates containing nuclear extracts should also be passed through a needle, sonicated, or treated with endonuclease to break down DNA and carbohydrates and reduce sample viscosity. Alternatively, protein samples may be purified and concentrated using TCA/acetone protein precipitation or buffer exchange.

Determining Protein Concentration

Following lysis, the samples should be filtered or centrifuged to remove insoluble cellular debris. The total protein concentrations of the samples are then determined to facilitate equal loading onto the gel for electrophoresis. Protein concentration is determined using biochemical colorimetric or fluorescent-based assays in combination with a spectrophotometer. The BCA assay is compatible with detergents and denaturing reagents, but cannot be used in conjunction with reducing agents. The Bradford assay is compatible with reducing agents, but cannot be used with detergents. The Lowry assay is highly precise, but is incompatible with detergents and reducing agents, and is a lengthier process [10].

Preparing Samples for PAGE

If the lysates will not be used immediately, store them in aliquots at -80°C. The protein samples are then appropriately diluted into sample buffer containing glycerol, to increase the sample density, and bromophenol blue, to observe migration of the sample through the gel. In addition, freshly added thiol reducing agents (DTT or β-mercaptoethanol) are typically used to reduce disulfide bonds and eliminate higher order structure in the protein samples. A standard sample buffer is 2X Laemmli buffer [1]. Alternatively, 4X or 6X recipes can be used to reduce dilution of the protein sample. The final protein concentration should be >0.5 µg/µl and between 3-5 µg/µl for optimum results. The protein sample can be concentrated through protein precipitation with TCA / acetone if necessary.

Gel Electrophoresis

The Principles of Electrophoresis

The standard technique used to separate proteins prior to immunoblotting is called discontinuous polyacrylamide gel electrophoresis (PAGE). Electrophoresis refers to the movement of charged particles in an electric field, and requires two electrodes of opposite charge (anode and cathode) separated by a conducting medium (the buffer). When charged molecules, such as proteins and DNA, are exposed to an electric field under these conditions, they migrate towards the electrode with their opposite charge [11]. The mobility of molecules in gel electrophoresis is dictated by the molecules charge, size, and shape, as well as the electrophoresis conditions. These include the ionic strength, pH, and composition of the buffer, the strength of the electric field, and the properties of the gel. A gel is used during protein electrophoresis to act as a size-selective sieve; facilitating protein separation based on molecular weight. The gel is typically mounted vertically between two buffer chambers and connected to electrodes. It is crucial that samples are loaded evenly across the gel, and that sample buffer is included in all wells, including those without sample, to ensure electrophoresis proceeds correctly.

Figure 2: The principle of electrophoresis.

When charged particles in a conducting medium are placed in an electric field, they will migrate towards the electrode with their opposing charge.

The Principle of Electrophoresis - Antibodies.com

The Purpose of SDS in SDS-PAGE

In the case of electrophoresis containing sodium dodecyl sulfate (SDS) and a reducing agent, also known as SDS-PAGE, proteins are separated based only on their mass. The anionic detergent SDS serves two roles: it denatures the proteins in the sample and it binds to their polypeptide backbones, providing them with a uniform negative charge and a similar charge to mass ratio (~1.4 g SDS binds to 1 g polypeptide). The reducing agent additionally breaks disulfide bonds in the proteins, further disrupting protein structure and imbuing the resulting polypeptides with a similar elongated shape. As all of the SDS-polypeptide complexes thus carry a negative charge, their migration in an electric field toward the positive electrode is limited by their size, where smaller molecules will migrate faster than larger ones.

Figure 3: Sodium dodecyl sulfate (SDS) coating a polypeptide.

SDS is added to a lysate sample to help denature proteins, as well as provide them with a uniform negative charge and similar 3D structure. A reducing agent, such as β-mercaptoethanol, reduces disulfide bonds, further disrupting the protein structure.

Sodium Dodecyl Sulfate (SDS) Coating a Polypeptide - Antibodies.com

The Properties of Polyacrylamide Gels

The properties of the gel also impact the mobility of the SDS-polypeptide complexes. PAGE gels are composed of an inert acrylamide polymer network with a defined pore size, and are made through the crosslinking of acrylamide and bisacrylmide upon the addition of the polymerizing agent ammonium persulfate (APS) and the catalyzing agent N,N,N’,N'-tetramethylenediamine (TEMED). The pore size can be adjusted by altering the polyacrylamide concentration in the gel (Table 4). Low percentage gels have large pores, allowing most proteins to pass through, and can therefore resolve high molecular weight proteins. In contrast, high percentage gels have small pores, severely restrict passage of large proteins, and are therefore better for resolving low molecular weight proteins. In addition, gradient gels provide a range of polyacrylamide concentrations within one gel, allowing proteins of varying size to be resolved simultaneously.

Figure 4: The chemical structure of a polyacrylamide matrix.

Polyacrylamide gels are made from crosslinked acrylamide and bisacrylamide following the addition of the polymerizing agent ammonium persulfate and the catalyzing agent TEMED.

The Chemical Structure of a Polyacrylamide Matrix - Antibodies.com

Table 4: Recommended gel percentages for different protein size ranges.

Protein Size Range Recommended Gel Percentage
4 – 40 kDa 20%
12 – 45 kDa 15%
10 – 70 kDa 12.5%
15 – 100 kDa 10%
25 – 100 kDa 7.5%

Other Forms of PAGE

In addition to SDS-PAGE, a nondenaturing form of electrophoresis called native-PAGE can also be used to separate proteins. Native-PAGE separates proteins based on their mass/charge ratio and shape, and can thus provide information about protein complexes. A third form of electrophoresis called two-dimensional PAGE (2D-PAGE) separates proteins along two dimensions. In the first dimension, they are separated based on their isoelectric point, the pH at which they carry no net charge, using isoelectric focusing. In the second dimension, they are separated by mass. This technique provides more detailed information for proteomics experiments.

Alternative Gel and Buffer Chemistries

Several distinct buffer compositions are used in SDS-PAGE, depending on the particular needs of an experiment. The most common system, described by Ulrich Laemmli [1] in one of the most widely cited papers of all time [12], is the inexpensive Tris-Glycine system. Tris-Glycine gels are compatible with SDS-PAGE and native-PAGE, and can resolve a wide range of proteins based on their size. The high alkalinity of Tris-Glycine gels, however, can lead to poor band resolution and can negatively impact downstream applications, such as mass spectrometry.

Bis-Tris gels, using either MES or MOPS buffer, are closer to neutral pH and reduce the risk of protein degradation and modification. As a result, this system produces sharper bands and is ideal for samples with low protein abundance and sensitive downstream applications, such as mass spectrometry and analysis of post-translational modifications. MES buffer is better suited for resolving small molecular weight proteins, while MOPS buffer can resolve medium molecular weight proteins [13].

Tris-Acetate gels are particularly suited for resolving high molecular weight proteins [14], while Tris-Tricine gels can resolve very low molecular weight proteins [15].

The Roles of the Stacking and Resolving Gels

Tris-Glycine SDS-PAGE is described as a “discontinuous” electrophoresis method because there are two distinct regions within the gel: the stacking gel and the resolving gel (Figure 5). The stacking gel has a lower concentration of acrylamide (4%), a lower pH (6.8 versus 8.8), and a unique ion composition compared to the resolving gel. The protein sample migrates within the stacking gel between a high mobility leading ion (Cl-) front and a low mobility trailing ion (glycine) front under the principles of isotachophoresis (ITP). As a result, the proteins “stack”, concentrating into a tight band before moving into the resolving gel. This promotes the formation of sharp protein bands and reduces protein aggregation. Once the proteins enter the resolving gel, the chemical properties of which more closely match those of the running buffer, the proteins “destack” and migrate according to the principles of zone electrophoresis. The electrophoretic mobility of the proteins thus depends on their mass, and the pore size of the gel creates a sieving effect.

Figure 5: Protein migration through a Tris-Glycine SDS-PAGE gel.

SDS-PAGE is considered a discontinuous electrophoresis technique, as it uses an inhomogeneous buffer system. These include the stacking gel, which consists of a low pH, low concentration acrylamide region that promotes concentration or “stacking” of the proteins in a sample, as well as the separating gel, which consists of a higher pH, higher concentration acrylamide region that separates the proteins according to size.

Protein Migration through a Tris-Glycine SDS-PAGE Gel - Antibodies.com

Hand-poured Gels versus Precast Gels

Precast gels are widely available in a range of chemistries, percentages, thicknesses, well numbers, and well volumes. While hand-poured gels may be advantageous for labs with unique needs, precast gels are convenient and can reduce experiment-to-experiment variability. This is especially important for quantitative experiments.

Protein Transfer

Transfer Methods

The proteins separated through gel electrophoresis are next transferred, or blotted, to a solid phase membrane. This process is required because proteins are more accessible to antibodies on the surface of these membranes and PAGE gels are difficult to handle once removed from their casts. The most common method of blotting is through the electrophoretic transfer of proteins from the gel onto the membrane, which is typically done through wet or semi-dry transfer. After transfer, a total protein stain, such as Ponceau S, can be used to visualize the efficiency of the transfer.

For wet electroblotting, the gel and membrane are sandwiched together, along with absorbent filter paper and sponges, and clamped into a cassette to prevent formation of air bubbles between the gel and membrane. The sandwich is submerged vertically in a conducting transfer buffer and exposed to an electric field. Under these conditions, the negatively charged proteins migrate out of the gel towards the positively charged electrode and become immobilized on the membrane. Wet electroblotting is a high efficiency transfer method that is well-suited for transferring low to high molecular weight proteins (>100 kDa), but is a time-consuming process, typically requiring ~1 hour – overnight.

Semi-dry electroblotting can be completed in 10 – 60 minutes. For this method, the gel and membrane are again sandwiched together with absorbent paper in a horizontal apparatus that is placed in direct contact with electrodes. Semi-dry blotting is better suited to lower molecular weight proteins (<300 kDa), as this technique exhibits reduced transfer efficiency.

Figure 6: The organization of a transfer sandwich for wet electroblotting.

During transfer, the negatively charged proteins will migrate out of the gel, toward the positively charged electrode, and onto the membrane.

The Organization of a Transfer Sandwich for Wet Electroblotting - Antibodies.com

Membrane Selection

The most common membranes used for immunoblotting are nitrocellulose and polyvinylidene difluoride (PVDF). Nitrocellulose can immobilize proteins and glycoproteins with high efficiency through hydrophobic interactions, but cannot be stripped and reprobed many times due to nitrocellulose’s fragility. Like nitrocellulose, PVDF has a high protein binding capacity, however, PVDF is highly hydrophobic and must be pre-wet with methanol prior to use. As it is less brittle than nitrocellulose, PVDF membranes can be reprobed many times. Both nitrocellulose and PVDF membranes are available in a range of pore sizes. While 0.45 um pore size membranes are generally applicable to most experiments, 0.1 – 0.2 um pore size membranes are more suitable for blotting low molecular weight proteins.

Transfer of Large Proteins

Protein transfer efficiency increases with transfer time and is lower for high molecular weight proteins. There are several ways to increase the transfer efficiency of these proteins. Transfer buffer does not usually contain SDS, as the residual SDS associated with the proteins following gel electrophoresis is sufficient to mediate their electrophoretic migration during transfer. However, the transfer efficiency for large proteins can be increased by adding SDS to the transfer buffer to a final concentration of 0.05 – 0.1% [16]. As methanol serves to remove SDS from proteins during transfer to increase protein immobilization on the membrane, the methanol concentration in the transfer buffer can also be decreased to <10%. In addition, the transfer process can be carried out overnight at 4°C to increase the transfer efficiency, however, smaller proteins can be lost if the transfer process proceeds too long.

Immunodetection

Blocking

As the solid phase membranes used in immunoblotting have a high affinity for proteins, antibodies applied to the membrane can adhere to it non-specifically. To prevent this background binding, and to improve the signal-to-noise ratio of the detection step, the membrane must be blocked prior to antibody incubation. The specific reagents used for blocking and the incubation time must be empirically determined. Common blocking agents include non-fat milk and bovine serum albumin (BSA). These can be solubilized in Tris-buffered saline (TBS) or phosphate-buffered saline (PBS), and 0.1% Tween-20 can be added to help prevent non-specific interactions. TBS in particular should be used if the detection method utilizes alkaline phosphatase (AP), as PBS can interfere with AP enzymatic activity. Sodium azide should be not used in buffers if a horseradish peroxidase (HRP) conjugated secondary antibody will be used, as azide inactivates HRP.

Immunodetection of Phosphorylated Proteins

If a membrane will be probed with a phospho-specific antibody, 5% BSA in TBST should be used for blocking. This is because milk contains high levels of the phosphoprotein casein, which will result in a high non-specific background signal if used in combination with a phospho-specific antibody. The results of immunodetection with a phospho-specific antibody should always be compared to an immunoblot showing the total protein levels.

Washing

Between antibody incubation steps, the membrane must be washed with TBS + 0.1% Tween 20 (TBST) or PBS + 0.1% Tween 20 (PBST) to remove unbound antibodies and to reduce the background signal. While too little washing can result in a high background signal, too much washing can reduce the signal-to-noise ratio.

Antibody Incubations

Not all primary antibodies can be used for immunoblotting, depending on the epitope they recognize. Refer to the antibody’s datasheet to ensure it is suitable for immunoblotting. The primary antibody is typically diluted into the wash buffer or the blocking buffer with a slightly decreased blocking agent percentage to help reduce non-specific binding. Although the optimum dilution of the antibody must be empirically determined, most antibody datasheets include recommended dilution ranges. The incubation can be carried out in a shallow box, in a tube, or in a heat-sealed plastic bag to decrease the volume of the primary antibody solution and minimize antibody use.

The proteins on a membrane are typically visualized through indirect detection by utilizing a labeled secondary antibody that recognizes the primary antibody based on its host species and isotype. This is in contrast to direct detection, which utilizes a labeled primary antibody. Indirect detection is usually more advantageous than direct detection. Multiple secondary antibodies bind to one primary antibody, leading to signal amplification. In addition, secondary antibodies are more versatile, as they can be used with many different primary antibodies.

Detection

The most common detection method uses enzyme-conjugated secondary antibodies in combination with a chemiluminescent, chromogenic, or fluorescent substrate. HRP-labeled secondary antibodies are widely available at low cost, are stable, and exhibit high enzymatic activity over a defined amount of time. AP- conjugated antibodies are also used to a lesser extent. Luminol and acridan-based substrates can be converted by HRP into chemiluminescent products, which can immediately be detected. Most chemiluminescent substrates are available with varying levels of sensitivity and can be used to enhance the signal in cases where the levels of the target protein are low.

Chemiluminescent substrates are compatible with digital imaging, which allows for quantitative western blotting. A CCD camera-based imaging system, enclosed in a dark cabinet, is typically used for this purpose. Fluorescently labeled secondary antibodies can provide a brighter signal and allow detection of more than one protein at a time but require expensive specialized imaging equipment.

Figure 7: Detection of target proteins using a chemiluminescent-based substrate.

A horseradish peroxidase (HRP)-conjugated secondary antibody will convert luminol in the presence of hydrogen peroxide (H2O2) to a chemiluminescent signal and a breakdown product (3-aminophthalate), mediating visualization of a target protein.

Detection of Target Proteins using a Chemiluminescent-based Substrate - Antibodies.com

Data Analysis and Quantitative Western Blot

Densitometry analysis of immunoblots can be performed using image-processing software. However, a number of factors must be taken into consideration when designing a quantitative immunoblot experiment. The proper amount of sample must be loaded on the gel to prevent signal saturation; this can be optimized by creating a standard curve of the protein loaded versus the band density for a target protein. An appropriate loading control must be used to compare samples across a blot. It is becoming more common to use total protein staining to normalize loading between samples, as opposed to using “housekeeping proteins” [17]. A number of detailed protocols for quantitative immunoblotting have been published [18-20].

Sample Protocols

Lysate from adherent culture cells

  1. Aspirate the culture medium from the cells, place the dish on ice, and wash twice with cold PBS.
  2. Add cold lysis buffer to the cells (1 ml / 107 cells in a 100 mm dish or 500 µl / 5x106 cells in a 60 mm dish).
  3. Scrape the cells from the bottom of the dish with a cold cell scraper and transfer the suspension to a cold microcentrifuge tube on ice.
  4. Continue lysis on ice with frequent agitation for 5 – 30 minutes. Alternatively, perform sonication or use a Dounce homogenizer to lyse the cells.
  5. Centrifuge the lysate at 4°C for 15 minutes at ~14,000 x g; the time and speed may vary depending on the cell type.
  6. Transfer the supernatant to a new tube on ice and discard the pellet.

Lysate from suspension culture cells

  1. Pellet cells in the culture medium at 2,000 x g for 5 minutes at 4°C.
  2. Aspirate the supernatant and wash the cells twice with cold PBS on ice.
  3. Resuspend the pelleted cells using lysis buffer.
  4. Continue with the lysis protocol for adherent cells from Step 4.

Lysate from tissue samples

  1. Dissect the tissue sample and immediately snap freeze in liquid nitrogen; store at -80°C for later use or proceed to the next step.
  2. Grind the sample in the presence of liquid nitrogen in a cooled mortar before transferring 1 g of the powdered tissue to a microcentrifuge tube on ice containing 1 ml cold lysis buffer. Alternatively, homogenize 1 g of tissue in 1 ml cold lysis buffer on ice until sufficiently disrupted.
  3. Continue lysis on ice with frequent agitation for 5 – 30 minutes.
  4. Centrifuge the lysate at 4°C for 30 minutes at ~35,000 x g.
  5. Transfer the supernatant to a new tube on ice and discard the pellet.
  1. Determine the total protein concentration for each lysate using BCA, Bradford, or Lowry assays, as appropriate.
  2. Dilute the lysates 1:1 with 2X Laemmli buffer with freshly added β-mercaptoethanol or DTT, and vortex thoroughly.
  3. Denature and reduce the samples by heating at 95°C for 5 minutes or 70°C for 10 minutes, and vortex thoroughly.
  1. Assemble the electrophoresis apparatus, insert the gel cassette, and fill both inner and outer chambers with running buffer according to the manufacturer’s instructions.
  2. Slowly load a molecular weight marker / ladder and the appropriate amount of sample(s) containing sample buffer to the wells of the gel. Use 20 – 30 µg of protein per well as a starting point.
  3. Connect the electrophoresis apparatus to the power supply and run according to the manufacturer’s recommendations. For the first 10 minutes of the run, use 5 – 10 V/cm of gel as the samples concentrate in the stacking gel. Once the dye front moves into the resolving gel, increase the voltage to ~100 V or as recommended by the manufacturer.
  4. Run the gel until the dye front runs off the bottom of the gel (~1 hour).
  1. Ensure the transfer buffer is pre-chilled at 4°C.
  2. Cut a piece of membrane and two pieces of filter paper to the size of the gel.
  3. If using a PVDF membrane, pre-wet the membrane in methanol for 2 minutes and then wash for 30 seconds in 1X transfer buffer. If using nitrocellulose, pre-treatment is not required. Carefully use tweezers to handle the membrane.
  4. Equilibrate the gel in transfer buffer for 10 minutes. To prevent the gel tearing, it may be easiest to move the gel into the transfer buffer while it is still attached to the glass cast / cassette.
  5. Soak the membrane, two pieces of absorbent filter paper, and two sponges in transfer buffer.
  6. Sequentially assemble the transfer sandwich in a shallow dish with transfer buffer, ensuring all components are wet. Start from the back, which will contact the negatively charged electrode (cathode). Layer a sponge, filter paper, the gel, the membrane, and a second piece of filter paper, building toward the positively charged (anode) electrode at the top.
  7. After sandwiching the gel and membrane between filter paper, use a roller or pipette to gently roll over the sandwich to remove air bubbles between the gel and membrane, which can impede transfer efficiency.
  8. Add the final sponge to the top of the stack and firmly lock the sandwich together.
  9. Fit the sandwich into the transfer cassette, ensuring the membrane side of the sandwich faces the anode and the gel side faces the cathode. Perform a wet or semi-dry transfer according to the manufacturer’s instructions. For wet transfer, run at 100 V for 1 – 2 hours or 30 V overnight at 4°C.
  1. Block the membrane in ~10 ml blocking buffer (5% BSA / TBST or 5% non-fat milk / TBST) in a small box for 1 – 2 hours at room temperature with gentle agitation.
  2. Incubate the membrane with the primary antibody diluted in TBST, with or without additional blocking agent, for 2 hours at room temperature or overnight at 4°C. Refer to the antibody’s datasheet for recommended dilution ranges. If using a heat-sealed plastic bag for the incubation, ~2 ml of primary antibody solution may be sufficient.
  3. Wash the membrane three times with 20 ml of TBST for five minutes each with gentle agitation.
  4. Incubate the membrane with the secondary antibody diluted in 10 ml TBST for 1 hour at room temperature. Refer to the antibody’s datasheet for the recommended dilution, but 1:5,000 is typical for HRP-conjugated secondary antibodies.
  5. Wash the membrane three times with ~20 ml of TBST for five minutes each with gentle agitation.
  6. If using an HRP-conjugated secondary antibody, use a chemiluminescent substrate kit for detection. Refer to the manufacturer’s instructions to prepare the substrate. Place the membrane on a plastic sheet, apply the substrate solution, and image the chemiluminescent signal with a CCD camera-based imaging system.

Mild Stripping

  1. Incubate the membrane in mild stripping buffer for 10 minutes with gentle agitation.
  2. Replace with new buffer and incubate for 10 minutes with gentle agitation.
  3. Wash with two changes of PBS for 10 minutes each.
  4. Wash with two changes of TBST for 10 minutes each.
  5. Proceed with blocking and antibody incubation steps.

Harsh Stripping

  1. Warm the harsh stripping buffer to 50°C in a fume hood.
  2. Incubate the membrane with the buffer for 45 minutes at 50°C with gentle agitation in a fume hood. Dispose of β-mercaptoethanol appropriately.
  3. Rinse the membrane under running water for 1 minute.
  4. Wash with three changes of TBST for 5 minutes each.
  5. Proceed with blocking and antibody incubation steps.

Troubleshooting Guide

Potential Issue Possible Solution
Post-translational modifications, including phosphorylation and glycosylation, can increase the apparent size of proteins on a blot. Use enzymatic treatments to remove a suspected modification and check the protein sequence for modification sites.
Alternative splicing can result in multiple protein isoforms of different sizes, and the expression of these isoforms may differ between different tissues, cell types, or culture conditions. Review the literature to determine if splicing variants are known to exist for the target protein.
Much larger bands than expected may correspond to multimers of the protein, suggesting that the sample was not fully reduced and denatured. Ensure disulfide bonds are reduced in the sample by adding fresh DTT or β-mercaptoethanol.

Ensure the sample is denatured by adding a chaotropic agent (such as urea).
Potential Issue Possible Solution
The transfer conditions were not appropriate for efficiently transferring large proteins. Include a small percentage (0.05%) of SDS in the transfer buffer.

Decrease the methanol concentration in the transfer buffer.

Perform the transfer at 30 V overnight at 4°C.
The gel used for electrophoresis was not appropriate for resolving large proteins. Determine if protein was left in the gel after transfer by staining the gel with a protein stain (such as Coomasie blue).

Use a low-percentage gel or gradient gel for resolving large proteins.

Use a different gel chemistry (ex. Tris-Acetate) for resolving large proteins.
Potential Issue Possible Solution
The protein samples may have been degraded during sample preparation. Always prepare samples on ice and include protease inhibitors in the lysis buffer.
Alternative splicing variants of the target protein may exist. Review the literature to determine if splicing variants are known to exist for the target protein.
The primary antibody may recognize other proteins with similar epitopes. Use a different primary antibody.
Potential Issue Possible Solution
The primary and / or secondary antibody concentration was too high or the conditions for incubation were incorrect (too long, at too high a temperature, etc.). Optimize the antibody incubations or try a different primary antibody.

The antibody concentrations / incubation conditions can be optimized by performing a dot blot checkerboard titration, where small volumes of sample are spotted directly onto a membrane and varying dilutions of an antibody are incubated with each spot.
Too much protein was loaded on the gel. Excess protein can lead to background signal; reduce the amount of sample loaded onto the gel.
The membrane was insufficiently washed. Increase the number of washes and / or the stringency of washes (increase the salt concentration of the TBST, change the buffer type from TBST to PBST, increase the Tween-20 concentration to 0.01 – 0.5%).

Alternatively, include Tween-20 in the antibody dilution buffers to reduce non-specific binding.
The membrane was insufficiently blocked. Use an alternate blocking agent, such as normal serum from the host species of the primary antibody.

Block overnight at 4°C.

Avoid using milk or BSA if the primary antibody was derived from a goat or sheep.
Excess SDS associated with the membrane bound non-specifically to the antibody. Wash the membrane after the transfer using wash buffer.
Potential Issue Possible Solution
The incorrect reagents were used at some point during immunoblotting (ex. the primary antibody host did not match the secondary antibody; the primary antibody was not suitable for western blot; the primary or secondary antibody was no longer active, etc.). Ensure that the correct reagents are used and are compatible with one another.
An improper blocking agent was used, such as milk for a phospho-specific antibody, resulting in the antibody non-specifically binding to the membrane. Use an alternate blocking agent, such as normal serum from the host species of the primary antibody.

Block overnight at 4°C.

Avoid using milk or BSA if the primary antibody was derived from a goat or sheep, avoid milk if using a phospho-specific antibody, and avoid PBS if using an alkaline phosphatase-conjugated secondary antibody.
The primary or secondary antibody concentrations were too low, the length of incubation time was too low, or the wash steps were too stringent. Optimize the antibody concentration by performing a dot blot checkerboard titration.

Reduce the detergent concentration and ionic strength of the washes, and perform the washes at 4°C.
The blot was stripped and reprobed, resulting in antigen removal from the membrane. Use a less stringent stripping procedure in the future.
The samples may have been over-transferred, under-transferred, or not transferred due to incorrect orientation of the transfer sandwich within the cassette. Use a total protein stain and the transfer of the molecular weight markers as indicators of transfer efficiency.

Optimize the transfer length and the transfer buffer composition (SDS, methanol concentration) for the size of the target protein.

Low molecular weight proteins can transfer through the membrane if the transfer proceeds for too long or at too high voltage. Use PVDF or small pore size nitrocellulose (0.2 um).
The target protein concentration was too low in the sample. Concentrate the protein samples using TCA / acetone precipitation, increase the amount of protein loaded on the gel, or increase the initial cell / tissue input during sample preparation if possible.
Potential Issue Possible Solution
The protein concentration differs between samples. During initial sample preparation, ensure that the same number of cells or amount of tissue is processed for each sample.

Once the protein is extracted from the samples, measure the concentration using an appropriate assay.
The wells were loaded unevenly or incorrectly. While loading the gel, make sure there is no spillover between wells and that the wells are evenly loaded.
The samples may express different amounts of the loading control protein. Check the total protein levels using a protein stain (such as Ponceau S) and / or use a different loading control.
Potential Issue Possible Solution
Too much sample was loaded onto the gel. Reduce the amount of sample loaded or dilute the samples.
The primary and / or secondary antibody concentration was too high. Optimize the antibody incubations by performing a dot blot checkerboard titration.
The substrate used for detection was too sensitive. Use a less sensitive substrate.
The exposure time during detection was too long. Perform the detection using multiple different lengths of exposure.
Potential Issue Possible Solution
The membrane and gel were not fully in contact during the transfer and / or bubbles were present between the membrane and gel. Remove air bubbles between the gel and membrane during assembly of the transfer sandwich by rolling over the sandwich with a pipette.

Ensure the transfer sandwich is not too loose when clamped shut, adding in additional filter paper or sponges if necessary.
The transfer buffer overheated, which caused bubbles to form between the gel and membrane. Use pre-chilled transfer buffer, include an icepack in the transfer chamber, reduce the voltage of the run, and / or perform the transfer at 4°C.
The membrane was allowed to dry out. Ensure the membrane is always wet, both during transfer and after the transfer.
Potential Issue Possible Solution
There was a problem during the electrophoresis step due to incomplete / heterogenous gel polymerization, overloading of the wells, incorrectly made running buffer, or incorrect running conditions. If using hand-poured gels, try repeating with a precast gel.

Reduce the amount of sample loaded onto the gel, or dilute the sample.

Ensure the running buffer and electrophoresis conditions are correct.
High salt concentration or differences in salt concentration between wells can result in band distortion. Reduce the amount of salt used during sample preparation.

Use TCA / acetone precipitation or buffer exchange during sample preparation.
The voltage was too high during gel electrophoresis, the running buffer became too hot, or the running buffer formulation was incorrect. Perform gel electrophoresis at a lower voltage for a longer time and / or perform electrophoresis at 4°C.

Ensure the running buffer was made correctly.
Potential Issue Possible Solution
The membrane was handled without gloves and oils were transferred to the membrane. Dirty tools (forceps, incubation boxes, etc.) were used to handle the membrane. Always use gloves to handle the membrane.

Always thoroughly clean tools and boxes that the membrane will come in contact with using ethanol.
The membrane was allowed to dry out. Ensure the membrane is always wet, both during transfer and after the transfer.
Potential Issue Possible Solution
The blocking solution was not sufficiently dissolved or should have been filtered to remove aggregates. Allow sufficient time to dissolve the blocking agent (milk powder) into buffer before blocking.

Alternatively, filter the blocking solution before use.
Potential Issue Possible Solution
The sponges used during gel electrophoresis were contaminated. Obtain fresh sponges.
The transfer buffer was contaminated. Prepare fresh transfer buffer and filter if necessary.

Buffers and Reagents

RIPA

  • 50 mM Tris, pH 8.0
  • 150 mM NaCl
  • 1.0% NP-40 or Triton X-100
  • 0.5% Sodium deoxycholate
  • 0.1% SDS (sodium dodecyl sulfate)

NP-40

  • 50 mM Tris, pH 8.0
  • 150 mM NaCl
  • 1.0% NP-40 (or Triton X-100)

Tris-HCl

  • 20 mM Tris-HCl, pH 7.5

Tris-Triton

  • 10 mM Tris, pH 7.4
  • 100 mM NaCl
  • 1 mM EDTA
  • 1 mM EGTA
  • 1% Triton X-100
  • Glycerol
  • 0.1% SDS
  • 0.5% Deoxycholate

2X Laemmli Sample Buffer

  1. 0.125 M Tris-HCl
  2. 4% SDS
  3. 5% β-mercaptoethanol
  4. 20% Glycerol
  5. 0.004% Bromophenol blue
  6. pH to 6.8
Component Stacking Gel 4% Resolving Gel 7.5% Resolving Gel 12%
30% Acrylamide / Bisacrylamide 1.98 ml 3.75 ml 6.0 ml
0.5M Tris-HCl pH 6.8 3.78 ml -- --
1.5M Tris-HCl pH 8.8 -- 3.75 ml 3.75 ml
10% SDS 150 µl 150 µl 150 µl
diH2O 9 ml 9 ml 5.03 ml
TEMED 15 µl 15 µl 7.5 µl
10% APS 75 µl 75 µl 75 µl
Total Volume 15 ml 15 ml 15 ml
  1. Prepare fresh APS and clean the glass plates of the gel cast with ethanol.
  2. Prepare the stacking and resolving gel solutions without TEMED and APS and assemble the gel casting system. Tip: You can check for leakage within your cast by filling the cast with diH2O first.
  3. Add TEMED and APS to the resolving gel solution, mix quickly but thoroughly with a pipette, and add to the cast to the appropriate level.
  4. Immediately overlay the resolving gel with isopropanol to ensure the gel is level, and allow polymerization to proceed for 15 – 30 minutes.
  5. Rinse away the isopropanol with diH2O and gently dry the area above the resolving gel with filter paper.
  6. Add TEMED and APS to the stacking gel solution, mix quickly, and add to the cast above the resolving gel.
  7. Place a comb gently into the solution, avoiding the formation of bubbles.
  8. Allow polymerization to proceed for 30 – 45 minutes.
  9. Gently remove the comb by pulling it straight upwards and rinse the wells with diH2O.

Running buffer (Tris-Glycine / SDS)

  • 25 mM Tris base
  • 190 mM Glycine
  • 0.1% SDS
  • pH to 8.3

Transfer buffer (Wet)

  • 25 mM Tris base
  • 190 mM Glycine
  • 20% Methanol
  • pH to 8.3

Transfer buffer (Semi-dry)

  • 48 mM Tris
  • 39 mM Glycine
  • 20% Methanol
  • 0.04% SDS
  • 24 g Tris base
  • 88 g NaCl
  • pH to 7.6 with HCl
  • H2O to 1 L
  • 100 ml 10X TBS
  • 900 ml H2O
  • 1 ml Tween-20

5% Milk Blocking Buffer

  • 5% w/v non-fat milk
  • 1X TBST

5% BSA Blocking Buffer

  • 5% w/v BSA
  • 1X TBST

Mild Stripping Buffer (1 L)

  • 15 g Glycine
  • 1 g SDS
  • 10 ml Tween-20
  • H2O to 1 L
  • pH to 2.2 with HCl

Harsh Stripping Buffer (100 ml)

  • 12.5 ml 0.5M Tris-HCl pH 6.8
  • 20 ml 10% SDS
  • 0.8 ml β-mercaptoethanol
  • H2O to 100 ml

References

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  3. Southern, E. M. Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis. J. Mol. Biol. 98, 503–517 (1975).
  4. Alwine, J. C., Kemp, D. J. & Stark, G. R. Method for Detection of Specific RNAs in Agarose Gels by Transfer to Diazobenzyloxymethyl-Paper and Hybridization With DNA Probes. Proc Natl Acad Sci U S A 74, 5350–5354 (1977).
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  8. Georgatos, S. D. & Blobel, G. Lamin B Constitutes an Intermediate Filament Attachment Site at the Nuclear Envelope. J. Cell Biol. 105, 117–125 (1987).
  9. Szymanski, E. P. & Kerscher, O. Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-Translational Modifications. J Vis Exp e50921 (2013). doi:10.3791/50921
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  14. Cubillos-Rojas, M. et al. Tris-acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass. Methods Mol. Biol. 869, 205–213 (2012).
  15. Schägger, H. & Jagow, von, G. Tricine-sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range From 1 to 100 kDa. Anal Biochem 166, 368–379 (1987).
  16. Bolt, M. W. & Mahoney, P. A. High-efficiency Blotting of Proteins of Diverse Sizes Following Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Anal Biochem 247, 185–192 (1997).
  17. Moritz, C. P. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics 17, 1600189 (2017).
  18. Taylor, S. C., Berkelman, T., Yadav, G. & Hammond, M. A Defined Methodology for Reliable Quantification of Western Blot Data. Mol. Biotechnol. 55, 217–226 (2013).
  19. Bass, J. J. et al. An Overview of Technical Considerations for Western Blotting Applications to Physiological Research. Scand J Med Sci Sports 27, 4–25 (2017).
  20. Butler, T. A. J., Paul, J. W., Chan, E.-C., Smith, R. & Tolosa, J. M. Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures. Biomed Res Int 2019, 5214821–15 (2019).
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