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Need to strip the membran before probe the internal control protein


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3 replies to this topic

#1 CarrieX

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Posted 31 December 2013 - 07:08 AM

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? 

 



#2 GNANA

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Posted 31 December 2013 - 07:24 AM

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 would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 jerryshelly1

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Posted 31 December 2013 - 09:23 AM

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. 



#4 bob1

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Posted 03 January 2014 - 02:28 PM

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.






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