I've been trying to optimize my western blotting protocol and recently started using the mini blot module from life. I ran my blot at 20V for 60 minutes as suggested by the manual and set up a second blank gel underneath the PVDF membrane to check for protein blow through. After blotting I stained both gels and the results are attached (sorry for the torn gels and blotchy photo).
Does anyone have any suggestions on how to decrease the amount of protein left on my original gel without adding to the blow through? My proteins of interest are between 40 and 100 kDa. Thank you for the help!
First questions first, why are you catching your blow-through on another gel and not another membrane? Two gels will alter the current balance across the gel-membrane sandwich and may effect your protein mobility.
I found this technique suggested in a protocol somewhere to test for blow-through, but that's a good point about the transfer conditions being altered. I'll repeat again with an extra membrane instead.
Hello Madeline Lewis ! Did you find the solution of incomplete protein transfer to PVDF membrane? I am facing the same problem with Mini blot module at 20V for 60 min.
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