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Need to strip the membran before probe the internal control protein - (Dec/31/2013 )

Hello! I am new in the proteomic area. I need to do a quantitative western blot and use Actin as internal control. I am not sure how to blot probe two proteins in one membrane (my target protein and Actin). Can I  cut out the area for Actin (~50kDa) and probe anti-Actin antibody and probe the other area with my target antibody? Or do I need to first probe my target protein and strip and reprobe the Actin? 



If the target protein and actin are far enough to be able to cut and probe, that would always be my choice instead of striping.


I am always nervous cutting blots and actin antibodies show such low cross-reactivity that I always order my experimental proteins with different conjugations to my internal control. This allows me to blot for both proteins simultaneously and if you are using a fluorescent secondary, it makes a beautiful blot. Maybe consider using a biotinylated antibody control in the future so you know there is almost zero crossreactivity. 


I agree with jerryshelly1 - so long as your two proteins are sufficiently separated, probe for the actin without stripping or cutting the blot.  I do this as standard using normal HRP based secondaries and x-ray film and very rarely have problems separating 50 kDa proteins from actin at 46 kDa.  It helps if you can use a ladder which shows up when probed, such as MagicMark from Invitrogen, but it isn't essential.