Western blotting involves 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. Below is our troubleshooting guide to help solve any issues that might be encountered with western blotting experiment.
| 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 µm). |
| 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. |