Weird IP - Please help (May/07/2008 )
I'm new to this forum and to IP. Anyway, I'm trying to determine whether my protein of interest interacts with each other, thus performed IP.
The protocol i used is:
(1) Collect cells into pellet
(2) Lysed cells with NP-40 Lysis Buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP-40 and 1 x PI)
(3) Spin to remove cell debrie in cold, transfer to new tube
(4) Quantitate protein level with Biorad Bradford reagent (around 1 mg)
(5) Add antibodies and incubate at a rotating platform in cold room (4 degrees) overnight
(6) Add 50 uL of protein G slurry to lysate
(7) Incubate at a rotating platform in cold room (4 degrees) for 3 hours
(8) Spin, remove supernatent, seperate proteins from beads, ran gel, transfer and add antibodies.
Ok guys.. so the weird part is I use A to pull B out.. and I can detect B but not A!!!
Does that means that the B i detected is not B?? (its a smear, and its slightly below the expected band size. need help regarding this as well.)
Is there anything wrong with my protocol??
All help will be greatly appreciated
There are several explanations, use the ones which suit your particular situation
1. A did not transfer to the membrane.
2. A did not migrate from the well because of its big size.
3. A is a membrane protein, and you boiled the beads.
4. A is too big or too small in Kd (did not run, did not transfer OR ran out, transferred out, respectively).
5. Antibody to A recognizes native A in the IP, but does not recognize denatured A in the western membrane.
6. B you detected may not be B. You need positive and negative controls to determine this.
7. If you don't think through and be careful about each step, many things may go wrong, such as wrong antibody, wrong combination of primary and secondary etc.
Hmm.. Seems like there's a lot of optimizing to do and I'm running out of time!!!
anyway, I think its possible that my A did not get transfer to my membrane as the kDa is almost 300. Going to rerun and stain gel with comassie just to make sure.
thanks for your help.