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 email@example.com.
|1||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.|
|2||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:|
|3||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.
|4||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.
|5||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.
|6||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.
|7||How much protein should I analyse?|
|8||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.|
|9||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.|
|10||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.|
|11||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.|
|12||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.|
|13||Why do I see a weak signal or no signal at all on my western blot?|
|14||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.|
|15||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