I have a flag-tagged protein and want to be sure it is properly expressing in my cells. When I assess by IF, I see clear nuclear enrichment (expected). But when I do a western, there is only a non-specific smear.
I am using sigma monoclonal anti-flag, and the western conditions work well for other flag-tagged proteins (shown by the band in lane 1). There is not an available endogenous antibody, which is why I am tagging the protein.
The nuclear extract was made by using pretty standard hypotonic buffers. Do you think the protein is just being denatured due to the tag, or the problem lies within the western? Any advice would be much appreciated, as I am not comfortable using just the IF to ensure the protein is being properly expressed. thank you!!!
While you do not mention your WB conditions, it is possible you need to use a lysis buffer for uniform cell lysates as hypotonic can be partial in breaking cells. Use a detergent based lysis buffer or Urea in sample buffer to denature your lysate. Alternatively pull down (immunoprecipitate) with anti- Flag then do a western to concentrate your tagged protein.
Does your protein interact with DNA? It simply could be stuck on it due to the hypotonic conditions. Maybe extracting with high salt helps.
Have you tried to lyse your cells with PBS-Tween 20 or -TX100 (leaving the nuclei intact) and then disrupting the nuclei by shearing (possibly directly in Laemmli Buffer)?
Wolfgang Schechinger Ah interesting, the protein is a transcription factor. So yes, I will try both of these recommendations. Thanks!
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