Please suggest if 15% gel for SDS-PAGE would be suitable or any other option? Secondly, for transfer, a nitrocellulose membrane 0.2um or PVDF? Any other tried and tested strategies will be appreciated.
Thanks in advance.
Hi, 15% gel should be fine but I would recommend 10-18% gradient gel as bends of small proteins tend to stretch in high-density gels. This way you will get much nicer blots. You should test both membranes as I noticed some antibodies blot preferentially better on different membranes. For example, ubiquitin blots are much stronger on PVDF membranes but histone proteins blot better on nitrocellulose (with antibodies I used).
I would recommend 0.22um pore PVDF with a shorter than normal transfer and possibly lower voltage. You may need to optimize the transfer conditions. I also agree that a gradient gel would be best for this application.
Thank you all 😊. Last time for transfer I used 0.2um nitrocellulose membrane at 100V for 1hr but couldn't detect the protein of interest. I think I should run it at 70-80V for longer time. Would it help?
12-20% gradient gel; make sure you have an appropriate prestained protein ladder with min mole wt of 5 or 10 kDa if your protein of interest is 8.5 kDa.
Keep on eye on the SDS-PAGE while it runs so that the markers (lowest) do not run off the gel before the run is complete.
transfer for 2 hrs at 80V to a PVDF membrane
carried out in ice cold buffer with sufficient ice packs to maintain the temperature
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