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CHOP protein western blot...help reqd!


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#1 uttess

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Posted 30 April 2011 - 02:34 PM

Hello, I am trying to look for CHOP protein using SDS page...Is anyone working with this protein and could help me out with the transfer conditions as I get very weak bands using semi wet transfer at 18 V for 90 minutes on PVDF membrane...plz help!

#2 kristoff

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Posted 03 May 2011 - 12:27 PM

First of all, you have to give more details. Like what kind of PVDF you use, transfer buffer composition, and also sds-page electrophoresis buffer and gel percentage.

If I were you I would try a PVDF with 0,2um pore size - it has higher binding capacity and it is less likely for small proteins to blow-through the membrane. Also, I alwyas use constant amperage (0.8 mA per square cm of transfer), and transfer for 1hr. Check your membrane with Ponceau S before you start with antibody incubations.

As for the sds-page conditions if you are happy with the resolution of don't change anything, but if you wanted to improve it, you can try the tris-tricine system with a 12% acrylamide gel. It works wonders for anything less than 30kD, down to even a few kD.

As soon as you give more details about your conditions, I can try to help you more.

Wish you luck!
K.

#3 uttess

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Posted 06 May 2011 - 03:20 PM

thnx for help...i use PVDF 0.2um memb..the transfer buffer contains 10% methanol and 5% transfer buffr diluted in dd water...i use invitrogen semi wet transfer device, as for the gels..it is 4-12% bis-tris gels...the problem is i do not get clear bands, although i can see faint bands but with high background. if i do not use this voltage then i do not see bands at all, this makes me think that something is going on with transfer

wont staining with ponceaus s before addition f antibody disrupt the membrane forever?

#4 mdfenko

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Posted 09 May 2011 - 07:56 AM

ponceau s is water soluble. after staining it is washed off with copious amounts of water (a bunch of changes). it is commonly used to determine the presence of proteins on the membrane and does not interfere with immunostaining.
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