Western Blot FAQs

The following are frequently asked questions (FAQs) in the Western Blotting (WB). If you have any issues in your Western Blotting, please feel free to contact support@sinobiological.com.

Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?

Check the blocking solution that you are using. Non-fat dry milk suit for all blots except phosphor tyrosine blots, which has Phosphorylated proteins that bind to the membrane. By blocking with this reagent, potential epitopes are present all over the blot. Diluting the anti-phosphor tyrosine antibody in this solution will occupy many of the epitopes, leaving fewer antibodies available for detection. We suggest to use 1% BSA in TBS + Tween 20 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:

Possible cause Solution
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. • Prepare fresh samples and avoid repeated freeze-thawing.
Antibody was contaminated. • Spin down vial and check for precipitate.
Second step reagents are not working. • Change second step reagents.
Samples degraded in storage or from repeated freeze-thawing.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.

How do I avoid "patchy", uneven spots appearing all over blot?

To avoid "patchy", or uneven spots appearing all over blot, try:
• Check all buffers used for bacterial contamination.
• Make sure membrane is fully immersed during antibody and washing incubations.
• Tease out all air bubbles between membrane and gel before performing transfer.
• Ensure uniform access to whole blot by placing membrane on a rocker/shaker.
• Ensure all western blotting equipment is properly washed.
• Filter HRP conjugate to remove any possible aggregates.
• Reduce substrate exposure time.

What can I use as positive control lysates for sinobiological WB antibodies?

To get the best Western Blotting performance from your Western blotting antibody, please use the positive control lysate recommended on our Technical Data Sheets. The positive control will help you duplicate our established results in initial experiments by precisely following our established protocols.
We recommend these lysates to be stored at -20°C for long-term storage. Lysates are extremely stable since they are 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 12 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 at -20°C.

How much protein should I analyse?

We recommend:
Sample type Per lane
whole cell lysate 50 μg
nuclear extract 25 μg

What percentage acrylamide gel should I use to resolve the proteins?

To best resolve your protein of interest, the percentage of acrylamide gel should be based on molecular weight.

Protein size (kDa) Acrylamide gel percentage (%)
<13 50
14-15 12
50 - 100 10
>100 8

What membrane should I use in a western blot?

We recommend using nitrocellulose. Although PVDF and other similar membranes can be used, they can sometimes lead to increased background particularly with goat polyclonal preparations.

What blocking buffer should I use?

We recommend using 1X TBS, 5% milk, 0.05% Tween-20. Blocking should be conducted for 30-60 minutes at room temperature or overnight at 4℃. Please note that Tween should be omitted from the buffer if incubating overnight.

What dilution of primary antibody should I use?

Check the datasheet of the individual antibody as this usually notes starting dilutions. If there is not specific information on the datasheet then we suggest that you perform a serial titration against relevant positive and negative controls.

Why is the actual western blot band size different from the predicted?

Western blotting is a technique that separates proteins by size. In general, the smaller the protein is, the faster it migrates through the SDS-page gel. However, the migration speed is also affected by other factors, so the actual band size observed may differ from that predicted.

Possible reason Notes
Post-translational modification e.g. phosphorylation, glycosylation etc., which increases the size of the protein.
Post-translational cleavage Most proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases.
Splice variants Alternative splicing may create different sized proteins transcribed from the same gene. These splice variants can lead to the production of different sized protein products. Expression of splice variants is highly variable depending on the specific tissue and experimental conditions being used.
Isoforms Many proteins express multiple isoforms that are different sizes. Expression of different isoforms for the same target protein is highly variable depending on the specific tissue and experimental conditions being used. Several web sites can be accessed to determine isoforms for specific targets.
Relative charge The composition of amino acids (charged vs non-charged).
Multimers e.g. Dimerization of a protein. This is usually prevented in reducing conditions, although interactions can result in the appearance of higher bands.

Why do I see a weak signal or no signal at all on my western blot?

Possible reason Notes
Antibody titration A titration experiment should be tested with multiple antibody concentrations to determine the appropriate antibody concentrations for optimal signal/noise ratios. In general this titration should range from 0.2 to 5.0 ug/ml.
Tissue/cell specificity Target protein expression is dependent on the cell or tissue being examined. A literature retrieve should be conducted to ensure that the system that you are using is appropriate for detecting the target protein.
Reconstitution Care should be taken in re-suspending lyophilized antibodies in order to ensure that the antibody is appropriately dissolved. Although the lyophilized antibody pellet is often at the bottom of the tube, in some cases the powder may have localized on the tube wall. Consequently, the dissoliution should cover the entire surface of the tube to ensure that the antibody is completely dissolved. The tube should then be briefly centrifuged to ensure that the entire powder is collected at the bottom of the tube.
Positive control The addition of a positive control lane to your western blot assay is the optimal means of evaluating whether the antibody is functioning appropriately and the experimental conditions are appropriate. Alternatively, a positive control based on the literature or internal experimental results can be used to assess whether the antibody is functioning appropriately.

Will this antibody react with the same target in a different species?

Primary species reactivity for all sinobiological antibodies is determined by western blot analysis. This is primarily done using a cell line or tissue derived from the primary species. The primary species reactivity as well as the validating tissues used are listed on the data sheets for each antibody that can be found on our web site.

Do my transfer conditions change if my protein of interest if >180kD?

• Transfer conditions/buffers need to be optimized for every lab
• Using 20% MeOH
• Adding 0.05% SDS
• Transfer times and power settings; try 1A for 1hr.
• Increasing lysate amounts will increase the number of copies to be transferred to your membrane