All of our antibodies undergo strict QC analysis by western blotting. We purify each lot with an affinity column, and we use the same protocol and conditions throughout the analysis. Also, we only release our antibodies after we obtain satisfactory results in comparison with the previous lot. If you do not obtain a signal, there could be a fundamental issue with your antibody or technique.
• You may not have used enough primary or secondary antibody. Please follow the recommended dilutions for the antibodies or test different antibody dilutions to determine the optimal antibody concentrations.
• There’s the possibility that your primary and secondary antibodies do not work together. Ensure that the secondary antibody was raised against the animal that the primary antibody was raised in.
• The NaN3, present in the washing buffer and secondary antibody solution, can sometimes cause problems.
• Your protein of interest may not be present or could be present at very low levels in the sample. Alternatively, the primary antibody may not recognize the protein from the species that you’re testing. If you are certain the protein is expressed, you can try an enrichment step to improve the signal. You should also check the antibody datasheet to ensure that the antibody cross-reacts with the species you’re using.
• Try loading more protein into your gels or use an alternative protein extraction method for sample preparation. Remember, the optimal sample preparation method for your target protein may not necessarily be identical to the one used for a control housekeeping protein (e.g., GAPDH).
The expected molecular weight (MW) listed in the product datasheet is based solely on the size of the target protein’s amino acid sequence. It’s important to remember that many factors can affect the banding pattern of your western blot including: (1) the existence of a splice variant, (2) the quality of the loaded sample, (3) the protein extraction method, and (4) the protein transfer conditions. To achieve accurate results, you may need to systematically adjust the protein extraction method or the protein transfer conditions.
• If the band’s MW is below the expected MW it could be due to a splice variant with a slightly different MW, or the target protein may have undergone proteolytic cleavage, generating a lower MW band. Changing your protein extraction method or transfer conditions may resolve this issue. You can also try the following steps:
o Heat the samples at 70°C for 10 min (instead of 100°C for 5 min) as this reduces potential degradation.
o Increase the transfer time. This will increase the transfer efficiency of high molecular weight (HMW) proteins. We recommend 100 mA for 20 h at 4°C.
• If the band’s MW is above the expected MW, it could be due to post-translational modifications (e.g., glycosylation or phosphorylation) or the existence of a different splice variant.
• In either event, you should use a blocking peptide as a negative control. This peptide is the antigen used for immunization and specifically blocks the antibody. So, in the presence of the blocking peptide, specific staining will disappear while non-specific staining will remain. You should use the blocking peptide at the ratio indicated in the datasheet, or 1 µg of peptide per µg of antibody.
High background levels may be due to the following: (1) a sub-optimal primary antibody concentration, (2) the type of membrane, (3) the blocking conditions, or (4) non-specific binding between the antibodies and the blocking reagent.
• Titrate the primary antibody to obtain the optimal concentration. Check the datasheet for the optimal dilution, but we recommend 1:200 in most cases.
• You may consider changing to a nitrocellulose membrane. Compared with polyvinylidene difluoride (PVDF) membranes, a nitrocellulose membrane tends to give you less background noise but does lack some of the detection sensitivity of a PVDF membrane.
• Also, ensure that the membrane doesn’t dry out during the western blot.
• If you haven’t blocked for long enough, you can end up with a lot of background in your WB. We recommend an hour with 1–3% bovine serum albumin (BSA), but overnight at 4°C can also work.
• Try adding 0.1%–0.5% of Tween-20 to the primary antibody solution and washing buffer since this strengthens the signal and reduces non-specific staining.
Usually, too many bands indicate that the sample preparation method was not optimal and/or that the target protein was degraded during sample preparation.
• Multi-transmembrane proteins like such as calcium channels are usually vulnerable to degradation during sample preparation. The optimal sample preparation method can vary depending on the tissue (e.g., brain, adrenal gland, etc.) and/or species (e.g., mouse, human, etc.). For this reason, we normally recommend running different samples prepared by different protocols in parallel, to establish the optimal sample preparation method. Remember, the optimal sample preparation method for your target protein may not necessarily be identical to the one used for a control housekeeping protein (e.g., GAPDH).
• If you are analyzing HMW proteins, you may need to adjust the SDS-PAGE conditions and the transfer conditions.
• Additional bands may indicate that there is a splice variant or a truncated version of your target protein.
• You can try a more diluted primary antibody concentration and/or add up to 0.5% Tween-20 to the primary antibody solution.
To make sure you get optimal blocking of the primary antibody, incubate the antibody, in parallel, with and without the antigen (the antibody/antigen ratio is available in the certificate of analysis delivered with the antibody) in a small volume (500 µl of 1% BSA in PBS) for 1 hour at room temperature with rotation. After the incubation, dilute each vial to the appropriate working concentration in the desired buffer and apply the contents of each vial to each membrane for parallel experiments.
• We recommend that you use the blocking peptide at the ratio specified in the datasheet, or 1 µg of peptide per µg of antibody.
• If you’re using a fusion protein to block the antibody, be aware that fusion proteins are sometimes more delicate to handle than peptides. If the fusion protein was correctly reconstituted (by adding 100 µl of PBS) and handled (avoidance of repeat freezing and thawing), a more diluted antibody together with an increased amount of fusion protein may be necessary for complete blocking of the antibody. We normally recommend adding 3 µg of fusion protein for each µg of antibody, but sometimes you need a larger ratio depending on the sample.
These curved bands (sometimes called “smiling bands”) are likely due to excessively high voltage, which causes excessively rapid migration of the proteins. Smiling bands can also occur when the temperature is too high.
• Reduce the voltage and run the gel in a cooler room.
If you’re looking for a protocol, take a look at our detailed Western Blot Protocol.