Below are some common issues associated with the western blotting technique.
Why are there multiple bands on my blot?
Several variables may account for this:
- An excessive amount of lysate loaded onto the gel may cause extra bands. Using decreasing dilutions of lysate will help to determine optimal loading amounts. An increased wash cycle may also alleviate this problem.
- Lower molecular weight products may be due to protein degradation. We suggest preparing fresh samples by lysing cells/tissues according to our recommended protocol. Compare the signal of your experimental samples with the positive control.
- Non-specific bands may be due to the secondary antibody. We recommend running a no primary antibody control: verify the appropriate dilution of the secondary detection system, run the Western blot with no primary antibody, and probe only with the secondary detection system.
- Higher molecular weight products may be a result of post-translational protein modifications, including, but not limited to, glycosylation, myristylation, phosphorylation, and/or ubiquitination.
- Additionally, higher molecular weight bands may also be a result of protein aggregation that is not resolvable by SDS and boiling.
Why is there a weak signal or no signal at all?
Several variables may account for this:
- This may be due to inadequate transfer of proteins during electroblotting. Try staining the membrane with India Ink or Ponceau-S immediately after the transfer. This is an effective and non-interfering method to verify proper protein transfer. Consult our recommended Western blotting protocols to optimize these electrophoresis and transfer conditions.
- The detection enzyme may be inactivated. Sodium azide inactivates horseradish peroxidase (HRP) irreversibly, so do not include sodium azide in any HRP-labeled reagents. Bacterial contamination also diminishes HRP activity. HRP conjugates should be kept bacteria-free, handled with sterile technique and stored under recommended conditions.
- The polyvinyl wrap from certain sources may quench the signal. We suggest repeating the incubation with chemiluminescence reagents and placing the blot between two pieces of write-on acetate transparency film, and then expose the film.
- The expression of the protein of interest may be very low. Sensitivity may be increased by performing an immunoprecipitation prior to the Western blot.
- In cases of very weak Ag expression, eliminating Tween during primary Ab incubation may improve Ab binding.
- To better facilitate Ag-Ab interaction you could incubate primary Ab at 37°C with gentle incubation and further incubate it overnight at 4°C.
Why is there high background and/or diminished signal with anti-phosphotyrosine antibodies?
Check the blocking solution that you are using. Non-fat dry milk, which we recommend for all blots EXCEPT phosphotyrosine blots, has phosphate-containing proteins that bind to the membrane. By blocking with this reagent, potential epitopes are present all over the blot. Diluting the anti-phosphotyrosine antibody in this solution will occupy many of the antibody binding sites, leaving fewer antibodies available for detection. So, use 1% BSA in TBS + Tween as the blocking solution - do not use milk.
Why does my antibody activity diminish in Western Blot over time?
If you are using one of our antibodies for Western Blot analysis and the reactivity seems to be diminishing over time, some points to consider are:
- Different samples were used - cells in culture will change characteristics over time, so we suggest thawing fresh cells.
- Samples degraded in storage or from repeated freeze-thawing.
- Antibody was contaminated - spin down vial and check for precipitate.
- Second step reagents are not working.
- Protein transfer was inefficient - stain blot with India Ink after transfer to confirm transfer of protein from gel to blot. Please note that antibodies are very stable and are unlikely to "go off" over time when stored appropriately.
How do I overcome inadequate or poor transfer problems?
Check to determine the size of the protein. If the protein is > 180 kDa, then you need to optimize transfer conditions by:
- Using 20% MeOH.
- Adding 0.05% SDS transfer buffer.
- Increasing loading amounts of lysate.
- Transfer for 1 hour at 1 A.
How do I avoid "splotchy" or uneven Western Blots?
To avoid "splotchy" or uneven blots when performing a Western Blot analysis:
- Extend the blocking time to optimize blocking effectiveness.
- Extend wash cycles (this is particularly important for tissue homogenate samples).
- Make sure that milk powder is in solution completely before adding it to the blot.
- Spin the tube containing the antibody solution before removing some for use, in case of protein aggregates.
What can I use as positive control lysates for BD Transduction Laboratories™ antibodies?
To get the best Western Blotting performance from your BD Transduction Laboratories™ antibody, please use the positive control lysate recommended on our Technical Data Sheets. This will help save you time, sample and antibody. The positive control will help you duplicate our established results in initial experiments by precisely following our established protocols. For detailed protocols please see our website, bdbiosciences.com/support/resources/.
We recommend these lysates to be kept at -20°C for long-term storage. Lysates are extremely stable since they are a cell preparations in which all enzymes are denatured and inactivated. We have done extensive research and confirmed stability under different conditions. For example, the integrity and quality of most lysates were not affected by repeated freeze-thaw cycles, or storage at 25°C for up to 10 weeks. The lysates, however, started to show degradation by four weeks at 37°C. There may, however, be some slight variation in stability depending on the source of the lysate so we do strongly recommend that they are stored as suggested.
How do I store BD Transduction Laboratories™ Antibodies?
All BD Transduction Laboratories™ antibodies (except the agarose conjugates) should be stored at -20°C. The buffer contains 50% glycerol, which prevents freezing at -20°C, thus allowing for repeated use of the antibody without the deleterious effects caused by freeze/thaw cycles. Do not store at 4°C or -80°C.
Is it okay that I received my antibody at room temperature?
Our antibodies are tested to be stable for a minimum of 5 days at 37°C. Because we ship overnight, extreme ambient temperatures are not expected to affect the stability of the antibody.