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Effect of MeOH and acetic acid on primary-secondary complex


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#1 Josip Madunic

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Posted 13 December 2013 - 06:14 AM

I made a serious mistake and accidentaly poured Coomasie destain solution (5 % MetOH and 7 % acetic acid) instead of TBST on PVDFmembrane. My incubation with secondary antibody was over and was washing it with TBST. Coomasie destain was on the membranes for about 10 min.

 

Is there any chance my primary-secondary complex was left intact? I am still waiting for films to be developed...



#2 bob1

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Posted 13 December 2013 - 01:50 PM

The methanol will precipitate the proteins and the acetic acid will probably partially denature the antibodies.  I don't know if it will affect the membrane at all, and how useful the membrane will be if you re-probed it.



#3 mdfenko

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Posted 14 December 2013 - 04:29 PM

the acetic acid will denature, possibly reversibly, the proteins, including the tag on your secondary (hrp or ap?). it probably won't effect a reprobe.


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#4 Josip Madunic

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Posted 16 December 2013 - 04:18 AM

Thank you for the replies, gonna mild strip then re-probe






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