Western Blotting: Principle, Procedure, Results and Applications

Western blotting involves a series of laboratory steps in which a protein sample is processed to obtain a detectable protein signal. The procedure includes sample preparation, gel electrophoresis, membrane transfer, antibody incubation and signal detection. These steps are carried out in a particular order.

Cell lysates and tissue extracts are commonly used as the starting protein samples. They contain a mixture of different proteins. At first, the protein sample is prepared and loaded into a gel for gel electrophoresis. The separated proteins are then transferred onto a membrane. After transfer, the membrane is treated with antibodies and the protein bands are detected.

The detected protein bands are examined for their position and signal intensity. These provide information about the protein being studied.

Each step of the procedure is connected with the other steps. The preparation of protein sample, selection of suitable reagents and use of proper experimental controls can affect the reliability of the detected result.

What is Western Blotting?

Western blotting is a laboratory technique used for the detection and identification of a specific protein from a mixture of proteins by gel electrophoresis, transfer of the separated proteins onto a membrane and their detection using specific antibodies. It is also known as immunoblotting or protein immunoblotting.

The sample may contain a large number of different proteins. Western blotting can detect a particular protein from such complex protein mixtures. The detected protein appears as a band on the membrane. The position of the band provides information about the apparent molecular weight of the protein, while its intensity can be used for the relative estimation of protein amount.

Western blotting is mainly concerned with the analysis of proteins, not deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

It is different from simple gel electrophoresis. Gel electrophoresis only separates the proteins, whereas Western blotting also involves the immobilization of separated proteins onto a membrane and their detection by antibodies.

Objectives of Western Blotting

To detect and identify a specific protein from a complex mixture of proteins present in biological samples, such as cell lysates or tissue extracts.
To determine the approximate molecular weight of a protein by comparing its detected band with standard protein markers.
To compare the expression levels of a particular protein in different cells, tissues or experimental conditions. The relative amount of protein can be estimated from the band intensity after proper normalization.
To detect post-translational modifications of proteins, such as phosphorylation, using antibodies specific to the modified protein.
To study different forms of a protein, including protein isoforms and cleavage products, based on their apparent molecular weights.

Principle of Western Blotting

The principle of Western blotting is based on the electrophoretic separation of proteins, their transfer onto a membrane and detection of a specific protein by antigen-antibody reaction. The proteins are first separated in a gel according to their electrophoretic movement. These separated proteins are then immobilized onto a membrane where specific antibodies are used to detect the target protein. The bound antibodies are detected using a suitable labelled detection system.

Protein Separation by Gel Electrophoresis

The separation of proteins is commonly carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Sodium dodecyl sulfate (SDS) is an anionic detergent which denatures the proteins and binds to their polypeptide chains, giving them an approximately uniform negative charge in proportion to their molecular mass.

When an electric field is applied, these negatively charged proteins move towards the positive electrode through the polyacrylamide gel matrix. The gel acts as a molecular sieve. Smaller proteins move more easily through the gel pores and migrate faster than larger proteins. This results in the separation of proteins according to their approximate molecular weight.

Native gel electrophoresis can also be used, where proteins generally retain their native structure and their migration depends on the size, charge and shape of the protein.

Transfer of Proteins to a Membrane

The separated proteins are transferred from the gel onto a protein-binding membrane, generally made up of nitrocellulose or polyvinylidene difluoride (PVDF). This process is referred to as electroblotting or electrophoretic transfer.

During the transfer, an electric field causes the proteins to move out of the gel towards the membrane. The proteins bind to the membrane surface and remain immobilized. Their relative positions are largely maintained as they were separated in the gel.

The membrane provides a solid surface where the transferred proteins are accessible for antibody binding.

Antigen–Antibody Binding and Signal Detection

The detection of the target protein is based on the specific binding between an antigen and antibody. A primary antibody recognizes and binds to a specific antigenic site (epitope) present on the target protein.

In the conventional indirect detection method, a secondary antibody binds to the primary antibody. The secondary antibody is usually conjugated with an enzyme or fluorescent label which is used for the detection of bound antibodies.

In chemiluminescent detection, an enzyme such as horseradish peroxidase (HRP) catalyzes a chemical reaction with its substrate and produces light. Fluorescent labels emit light when excited by a suitable light source. In the colorimetric method, the enzyme reacts with a suitable substrate to form a coloured product.

The generated signal appears at the position of the target protein on the membrane, forming a detectable protein band. Some Western blotting methods also use directly labelled primary antibodies for detection without requiring a secondary antibody.

Materials Required for Western Blotting

The following instruments, equipment and reagents are required for Western blotting.

Instruments and Equipment

Electrophoresis Apparatus – The apparatus consists of a gel tank with electrodes. It is used for the separation of proteins by gel electrophoresis.
Power Supply – It provides the electric current required for gel electrophoresis and electrophoretic transfer of proteins.
Gel Casting System – The gel casting system is made up of glass plates, spacers, casting stand and combs which are used for preparing polyacrylamide gels. Precast gels can also be used.
Protein Transfer Apparatus – The separated proteins are transferred onto a membrane using this apparatus. It may be a wet transfer tank or a semi-dry transfer system.
Micropipettes and Tips – Used for measuring and transferring small volumes of protein samples, buffers and other reagents.
Centrifuge – A microcentrifuge is used to separate the insoluble cell debris from the protein-containing solution during sample preparation.
Heating Equipment – A heating block or water bath is used for heating the protein samples when denaturation is required.
Shaker or Rocker – It provides gentle movement of the membrane during blocking, antibody incubation and washing.
Protein Detection System – The detection system depends on the type of signal produced. A chemiluminescence imaging system, fluorescence scanner or suitable colour detection system is used for recording the protein bands. Photographic films can also be used for chemiluminescent detection.
Spectrophotometer or Microplate Reader – These instruments are used to measure the protein concentration when a colorimetric protein assay is performed.

Reagents and Consumables

Protein Samples – Cell lysates, tissue extracts or other biological samples containing proteins are used as starting materials.
Lysis Buffer – It is used to break open the cells and extract the proteins. Commonly used buffers include radioimmunoprecipitation assay (RIPA) buffer and other suitable protein extraction buffers.
Protease and Phosphatase Inhibitors – Protease inhibitors are added to reduce the degradation of proteins during extraction. Phosphatase inhibitors are used when the phosphorylation state of a protein needs to be preserved.
Protein Loading Buffer – The loading buffer is mixed with the protein sample before electrophoresis. It commonly contains sodium dodecyl sulfate (SDS), glycerol, tracking dye and buffering agents for denaturing electrophoresis. Reducing agents may also be added.
Polyacrylamide Gel – It is used to separate the proteins according to their electrophoretic mobility. For manual gel preparation, acrylamide-bisacrylamide solution, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) are required.
Electrophoresis Running Buffer – The running buffer provides suitable pH and ions for the migration of proteins through the gel. Tris-glycine-SDS buffer is commonly used for conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Protein Molecular Weight Marker – It contains proteins of known molecular weights. These markers are used to determine the approximate molecular weight of the separated proteins.
Transfer Membrane – Nitrocellulose or polyvinylidene difluoride (PVDF) membrane is used for the immobilization of separated proteins.
Transfer Buffer – It provides the ionic medium required for transferring the proteins from the gel onto a membrane by electrophoretic transfer.
Blocking Solution – Non-fat dry milk, bovine serum albumin (BSA) or suitable commercial blocking buffers are used to block the unoccupied protein-binding sites on the membrane.
Primary Antibody – The primary antibody is specific to the target protein. It binds to the target protein present on the membrane.
Secondary Antibody – The secondary antibody binds to the primary antibody and is usually conjugated with an enzyme or fluorescent label. It is required for conventional indirect detection. A secondary antibody is not required when a directly labelled primary antibody is used.
Washing Buffer – Tris-buffered saline with Tween-20 (TBST) or phosphate-buffered saline with Tween-20 (PBST) is used to remove the unbound antibodies and other reagents.
Detection Reagents – Chemiluminescent or colorimetric substrates are used with suitable enzyme-labelled antibodies. For fluorescent detection, fluorescently labelled antibodies and a suitable imaging system are used.
Other Consumables – Microcentrifuge tubes, pipette tips, filter papers, absorbent pads, incubation trays and laboratory-grade water are used for handling the samples and membranes.

Procedure of Western Blotting

The common Western blotting procedure using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is carried out in the following steps.

Step 1 – Protein Extraction and Sample Preparation

Collect the cell or tissue sample and keep it cold during protein extraction.
Wash the cells with cold phosphate-buffered saline (PBS), if required, and add a suitable lysis buffer. For tissue sample, homogenize the tissue in lysis buffer.
Add protease inhibitors to prevent protein degradation. If phosphorylated proteins are being studied, phosphatase inhibitors are also added. Lysis is generally carried out on ice.
Centrifuge the lysate and separate the insoluble cell debris. Carefully collect the protein-containing supernatant in a clean tube. Membrane proteins, nuclear proteins and some other proteins may need special extraction buffer or additional extraction method.

Step 2 – Protein Quantification and Sample Preparation for Electrophoresis

Measure the protein concentration of each sample by a suitable assay such as bicinchoninic acid (BCA) assay or Bradford assay. The selected assay should be compatible with the lysis buffer.
From the measured concentration, take equal amount of total protein from each sample for electrophoresis.
Add loading buffer containing sodium dodecyl sulfate (SDS), tracking dye and other required components to the protein samples. For reducing SDS-PAGE, dithiothreitol (DTT) or beta-mercaptoethanol is also added.
Heat the samples for denaturation when required. Temperature and heating time depend on the protein. In non-reducing electrophoresis, the reducing agent is not added and heating condition may also be changed.

Step 3 – Protein Separation by SDS-PAGE

Prepare a polyacrylamide gel according to the expected molecular weight of the target protein. Precast gel can also be used.
Fit the gel into the electrophoresis apparatus and add the required running buffer.
Load the prepared protein samples into separate wells. Load a protein molecular weight marker in another well.
Apply the required voltage. The proteins migrate through the gel and are separated according to their apparent molecular weights. Continue electrophoresis until the tracking dye reaches the required position.

Step 4 – Transfer of Proteins onto a Membrane

Remove the gel after electrophoresis and prepare the transfer membrane. Polyvinylidene difluoride (PVDF) membrane is generally pre-wetted with methanol and then equilibrated in transfer buffer. Nitrocellulose membrane is soaked in transfer buffer.
Arrange sponge pads, filter papers, gel and membrane to make the transfer sandwich. Keep them soaked in transfer buffer and remove air bubbles between the gel and membrane.
Place the gel towards the negative electrode (cathode) and membrane towards the positive electrode (anode). The negatively charged proteins are transferred from the gel to the membrane.
Carry out protein transfer by a wet or semi-dry transfer system. Transfer condition depends on the equipment, membrane and size of the proteins.
The membrane can be stained with a reversible stain such as Ponceau S after transfer. This is used to check the protein transfer. Uneven staining or missing regions can indicate incomplete transfer.

Step 5 – Membrane Blocking

Remove the reversible stain by washing, if it was used. Place the membrane in a suitable blocking solution such as non-fat dry milk or bovine serum albumin (BSA).
Incubate the membrane in blocking solution, commonly for about 1 hour at room temperature with gentle shaking. It blocks the unoccupied protein-binding sites and reduces nonspecific antibody binding.
Select the blocker according to the target protein and antibody. BSA is often used for phosphorylated proteins because casein present in milk may interfere with some phospho-specific antibodies.

Step 6 – Primary Antibody Incubation

Dilute a validated primary antibody specific to the target protein in a suitable antibody incubation buffer.
Incubate the membrane with primary antibody, usually for about 1 hour at room temperature or overnight at 4°C. The actual incubation condition depends on antibody and experiment. Keep gentle shaking during incubation.
Wash the membrane several times with Tris-buffered saline containing Tween-20 (TBST) or another suitable washing buffer. This removes the unbound primary antibody.

Step 7 – Secondary Antibody Incubation

Select a secondary antibody that recognizes the host species of the primary antibody. It is commonly conjugated with horseradish peroxidase (HRP) or with a fluorescent label.
Incubate the membrane with diluted secondary antibody, commonly for about 1 hour at room temperature. Its concentration depends on the antibody recommendation and detection system.
Wash several times with TBST or another suitable washing buffer. Incomplete washing can produce high background signal.
When a directly labelled primary antibody is used, secondary antibody incubation is not required.

Step 8 – Signal Development and Visualization

Add a suitable detection reagent after washing. For HRP-based chemiluminescent detection, add the chemiluminescent substrate which produces light in the presence of the enzyme.
Record the emitted light using a chemiluminescence imaging system or suitable photographic film. Fluorescent Western blots are scanned with a fluorescence imaging system. A suitable enzyme substrate can also be used for colorimetric detection.
The target proteins appear as bands on the membrane image. Adjust the exposure to obtain clear bands without signal saturation.
Save the original blot image with imaging settings and sample details for further analysis. Keep the complete image and suitable control data.

Controls in Western Blotting

Controls are used in Western blotting to check the specificity of detected bands, differences in protein loading and transfer of proteins onto the membrane. Different controls examine different parts of the experiment. One control cannot replace the others.

Positive and Negative Controls

Positive control- A sample known to contain the target protein is included with the experimental samples. It should produce the expected protein band. The positive control indicates that the antibody and detection system are capable of detecting the target under the conditions used. It does not confirm that every band produced in the experimental samples represents the target protein.

Negative control- A sample in which the target protein is absent is used as negative control. A knockout cell, tissue or lysate is one of the stronger controls when available. The expected target band should disappear in the knockout sample. If a band remains at the same position, nonspecific antibody binding or cross-reactivity should be considered. Knockdown samples can also be used, but incomplete knockdown may still give a target signal.

A secondary-antibody-only control is carried out without the primary antibody. Any signal appearing in this control is produced by nonspecific binding of the secondary antibody or the detection system. It does not test the specificity of the primary antibody.

Antibody specificity can also be checked by comparing the signal obtained with different antibodies recognizing independent regions of the same target protein. The band should agree with the expected protein. Genetic loss of the target gives stronger evidence because the expected band can be compared directly between target-positive and target-negative samples.

Loading and Transfer Controls

A loading control is used to check differences in the amount of protein present between lanes. Housekeeping proteins such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin are commonly used for this purpose. Their expression must remain stable under the experimental conditions being studied. This should be verified. A housekeeping protein that changes after treatment, between tissues or between experimental groups cannot provide a reliable reference.
The signal of the housekeeping protein should also remain within its linear detection range. Highly abundant housekeeping proteins can become saturated even when the amount of loaded protein continues to increase. In such condition, an apparently similar housekeeping band does not necessarily indicate equal protein loading.
Total-protein staining provides another loading control by measuring the protein present throughout each lane rather than depending on one reference protein. Ponceau S staining and suitable stain-free methods can be used. It can show unequal loading and variation introduced during transfer. The selected total-protein method should have a suitable linear range for the protein amounts used.
Transfer control is used to check whether proteins have moved from the gel onto the membrane and whether the transfer is reasonably uniform. Staining the membrane for total protein after transfer can show the transferred protein pattern. The protein molecular weight marker and, where suitable, examination of the post-transfer gel can also help in checking transfer. Poor or uneven transfer can produce differences between lanes even when equal protein amounts were initially loaded.
Loading and transfer controls account for technical differences in the amount of protein detected in different lanes. They do not establish the specificity of the target antibody. Positive, negative and antibody-specificity controls are required for that purpose.

Results and Interpretation of Western Blotting

The result of Western blotting is observed as one or more protein bands on the membrane. The position of the band is compared with the protein molecular-weight marker, while the intensity of the band represents the amount of detected antibody-bound signal. Western blot results are generally interpreted together with positive, negative, loading and transfer controls.

Interpretation of Protein Band Patterns

Expected band- A band appearing near the expected molecular mass of the target protein (in kDa) supports the presence of that protein in the sample. The molecular-weight ladder is used to estimate its apparent molecular mass. A band at the expected position alone is not complete proof of protein identity. Antibody specificity and suitable controls are also required.
Positive result- A distinct band is detected at the expected position and the positive control also gives its expected band. The negative control should remain negative. This gives stronger evidence that the detected signal is associated with the target protein.
Negative result- No target band is detected in the sample. This can be interpreted as “not detected under the conditions used” when the positive control worked and the loading and transfer controls are satisfactory. It does not always mean that the protein is completely absent. A low amount of protein, poor antibody recognition or technical loss can also produce no detectable band.
Unexpected bands- One antibody may sometimes produce more than one band. A lower molecular-weight band can result from protein degradation or may represent a smaller protein form. Protein isoforms can also migrate differently. Bands at altered positions may occur with protein modifications or unusual migration, while nonspecific antibody binding can produce unrelated bands. These bands need additional validation before they are assigned to the target protein.
Weak band- A faint target band indicates a weak detected signal. It may occur due to a lower amount of target protein, but antibody binding, transfer, exposure and other experimental conditions also affect the signal. Band intensity alone should not be used to decide the cause.

Hypothetical Western Blot Result

The following is a hypothetical example where the expected target protein is approximately 50 kDa.

M = molecular-weight marker
P = positive control
A, B and C = experimental samples
N = negative control

The positive control (P) has a strong band at about 50 kDa. Sample A also contains a strong band at the same position. It would be considered positive for the target under this hypothetical experiment. A faint additional band is present near 75 kDa in sample A. This band cannot be assigned to the target only from its position.

Sample B has a weaker band at about 50 kDa. It gives a positive but weaker signal. Sample C has no detectable target band. The negative control also shows no band at 50 kDa.

If the loading and transfer controls are satisfactory, the blot would show a stronger target signal in A than B, while the target was not detected in C. The additional 75 kDa band in A would require further checking for nonspecific binding, a different protein form or another cause.

Band Intensity and Relative Protein Quantification

The darkness or fluorescence of a protein band can be measured by densitometry. A region containing the band is measured and the nearby or appropriate local background signal is subtracted. Background correction must be applied consistently because background itself can change the measured band value.

The target-band signal is then normalized against a suitable loading control or total-protein measurement. For example, a target band intensity of 80 units and a loading-control intensity of 40 units gives a normalized value of 2.0. Another sample having 40 and 40 units gives 1.0. Under comparable experimental conditions, the first sample has about a two-fold higher relative detected signal.

This comparison is valid only when the target and normalization signals are within their tested linear range. Saturated bands cannot be used reliably for densitometric comparison because an increase in protein may no longer produce a proportional increase in measured signal.

A visible band provides qualitative detection. Densitometry with proper normalization can provide relative or semi-quantitative comparison between samples. Measurement of an actual protein amount requires a validated quantitative assay with an appropriate calibration or standard curve containing known amounts of protein.

Applications of Western Blotting

Used to detect and identify a specific protein from a complex mixture of proteins.
Used to compare the relative amount or expression of a target protein between different samples.
Used to estimate the apparent molecular weight and detect different protein forms, isoforms or cleavage products.
Used to study post-translational modifications of proteins such as phosphorylation, ubiquitination and glycosylation.
Used to study protein-protein interactions when combined with suitable experimental methods.
Used to study the cellular or subcellular localization of a protein.
Used for checking and validating the specificity of antibodies against their target proteins.

Advantages of Western Blotting

It can specifically detect a particular protein from a complex mixture of proteins.
It provides information about the apparent molecular weight of the detected protein along with its presence.
It is sensitive and can detect low amounts of target protein when a suitable antibody and detection system are used.
It can be used for relative or semi-quantitative comparison of protein levels between different samples.
Different protein forms, cleavage products and some post-translationally modified proteins can also be distinguished when suitable antibodies are available.
The transferred proteins remain immobilized on the membrane and are easily accessible for antibody detection.
The same membrane can sometimes be stripped and reprobed for another protein, reducing the need to prepare a separate blot.

Limitations of Western Blotting

It depends greatly on the quality and specificity of antibodies. Cross-reactive antibodies can produce nonspecific bands.
Western blotting is generally semi-quantitative, and accurate comparison requires signals to remain within the linear detection range. Signal saturation can give inaccurate results.
The result can be affected by sample preparation, protein loading, transfer and detection conditions, reducing reproducibility between experiments.
It is a time-consuming technique involving several separate experimental steps.
Proteins present in very low amounts may be difficult to detect when the antibody or detection system has insufficient sensitivity.
Housekeeping proteins are not always expressed at a constant level. An unsuitable loading control can affect protein normalization.
Only a limited number of samples can be loaded on a single gel, restricting the number of samples compared in one blot.
A band at the expected molecular weight alone does not confirm the identity of a protein. Antibody specificity and suitable controls are also required.

Common Western Blot Problems and Troubleshooting

If no protein band is obtained, check the positive control first. Incorrect primary or secondary antibody, insufficient target protein and poor transfer can produce no signal. Antibody dilution and transfer conditions should be checked again.
A very faint band can result from low amount of target protein, highly diluted antibody or incomplete transfer. More sample can be loaded within a suitable range and the antibody concentration or transfer condition can be optimized.
High background is commonly produced by excess antibody, insufficient blocking or poor washing. Old buffers and very long exposure can also increase the background. Reduce the antibody concentration, increase washing and use fresh buffers.
Multiple or unexpected bands may occur due to nonspecific antibody binding or degradation of the target protein. Keep the sample cold and use protease inhibitors during extraction. Antibody concentration, blocking and washing conditions can also be changed, but the antibody should be validated before assigning any band to the target protein.
Smeared protein bands can be seen with poor sample quality, protein precipitation or excessive amount of sample. Reduce the protein load and check sample preparation before repeating the blot.
Blurry or distorted bands may occur when electrophoresis is carried out at too high voltage or when the gel is not suitable for separation of the target protein. Lower the running voltage and select a suitable polyacrylamide gel concentration.
Uneven or patchy signal is commonly associated with poor contact between the gel and membrane, trapped air bubbles or uneven incubation. Remove all bubbles while making the transfer sandwich and keep the membrane under gentle movement during antibody incubation and washing. Aggregated secondary antibody can also give irregular spots.
Poor transfer of high or low molecular-weight proteins requires adjustment of the transfer time, voltage or current and transfer buffer. Transfer condition should be optimized according to protein size and the membrane used.
Very dark, saturated or “ghost” bands can result from too much protein, high antibody concentration or excessive exposure. Reduce the amount of loaded protein, antibody concentration or exposure time.
Unequal signal between lanes may come from unequal protein loading or uneven transfer rather than a true biological difference. Check the total-protein pattern or a suitable loading control before comparing the target bands.

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