CHOP protein western blot...help reqd!
Posted 30 April 2011 - 02:34 PM
Posted 03 May 2011 - 12:27 PM
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!
Posted 06 May 2011 - 03:20 PM
wont staining with ponceaus s before addition f antibody disrupt the membrane forever?
Posted 09 May 2011 - 07:56 AM
genius does what it must
i do what i get paid to do