IP questions - Take a look at these recipies and see what you think (Jun/26/2007 )

I'm going to radiolabbell my stable expressing cell lines with 35S and try to do some IP's.
Like many things in science much depends on what you lyse your cells in and how good your antibody is.

I think I have a good antibody, based on Western Blot, but I am concerned about lysis methods. So I came up with three different recipies to try on my microsomal fraction of cellular protein.

1xTY
50mM Trish ph 7.4
150mM NaCl
1M KCl
2.5% CHAPS

1x RET
50mM Tris ph 7.4
100mM NaCl
0.05% SDS
0.5% Na deoxycholate
5uM EDTA
0.5% TritonX100
0.02% Na Azide

1x RIPA
50mM Tris pH 7.4
1% NP-40-
0.25% Na Deoxycholate
150mM NaCl
1mM EDTA

What I am going to do after a starve and pulse is collect my cells and make a microsomal pellet and then suspend them in each of these 3 buffers, incubate on ice 30 min, add 2 ug of my primary antibody and rock O/N at 4C. Then in the morning, add the Protein A and rock for 1hr r.t. Wash the pellet 5x with same buffer. Add Lemmli and DTT (100mM final conc) to the Protein A pellet , heat 95C 5 min then onto the gel.

Any comments or suggestions?

I'm thinking of doing each of these in duplicate with the buffers but to one set add DTT to 100mM. I'm curious as to what effect it will have on the Ab binding to the Protein A. It shouldn't be an issue with Ab-Ag interaction as that is in the Fab fragment.

Does anybody have any thoughts on using a Hepes buffers instead of a Tris buffer?