Can anyone help solve some troubles with co-immunoprecipitation? - (Jun/28/2014 )
I am doing co-immunoprecipitation to detect proteins interaction, the problem is that sometimes I see interaction between my two proteins and it's enhanced by phosphorylation and sometimes I can't see any interaction. I do exactly the same experience, same ripa buffer composition ( beads pre blocked with 5% BSA to get rid of non specific binding of beads to protein) but the result is not the same ( it works for one or two months and then nothing for two months, after some tests it work again and then nothing).
Also when it works, I do a reload to normalize my input I do not see what I saw in the first loading may be one freeze thaw is not good even though I see the immunoprecipitated protein
So, what can influence the interaction of two proteins? And how can i improve this interaction?
Can you provide details of your experimental procedure including details of your lysis buffer (especially with respect to protease and phosphatase inhibitors).
Freeze/thaw cycles can easily disrupt protein complexes.
Thank you for your reply,
I transfect 293T cells with protein A and Protein B (tagged with HA) plus or minus a kinase.I immunoprecipitate protein A and try to detect the tagged one.
my ripa buffer is 50mM tris, 0.7M NaCl, 2% NP40,0.5% deoxycholic acid, proteases inhibitors cocktail, PMSF, sodium orthovanadate, sodium fluoride.
I add RIPA buffer to the pellet, passe through a gauge needle suringe 10 times, centrifuge 10 min max speed, transfere supernatent to new eppendorf,
add andibody against protein A. At the same time i preblock my beads with 5% BSA (Centrifuge beads, add BSA o/n), at 4 degree.
the next day wash beads twice with ripa buffer, resuspend in PBS and add them to lysats+ Antibody 3h at 4 degree. Finally, i wash the beads 3 times 10min with rotation, add laemmli, boil 2 min and put at - 80 degree. The next day i do my WB. it seems that prot A and B interact together and this interaction is enhanced with phosphorylation. As i said i always see prot A the probleme is Prot B even i am doing exactely the same thing each time, somme times it work and some time times no, i don't know why and i am wasting my time by trying other conditions like RIPA with NaCl, or NP40, to use triton instead of np40 and other thing but no results. So I think that the conditions i am using are good since it worked before and the probleme is else where but i can't find it.
Make sure that when you spin down the beads that you use a low RCF (100-500 RCF) - high RCF can cause dissociation of the protein complexes from the beads.
Also make sure that when you do your washes, you dribble the wash down the side of the tube, then invert the tube gently to resuspend the beads. Rotation is unnecessary for washing steps. Washes shouldn't take more than 5 min each.
700 mM salt is quite high - 4.7 x physiological concentration - usually these sorts of things are done in the 150 (normal saline) -300 mM NaCl range. Deoxycholate is a quite strong detergent, try removing it and see if it improves your IP. 2% NP-40 (assuming you are actually using NP-40, not nonidet-p40) is also high - most protocols call for 0.5-1%
You might want to do a longer IP - try overnight with your lysate.
How much antibody are you using for each IP? What sort of antibody (mono? poly?)
I know that 700mM NaCl is more than what people use, but it worked for me. Now i have tried 150mM and 700mM side by side and any of them didn't work, same thing for the NP-40, i used igepal wich is the substitute of nonidet 40) i used 1% at the begining but since the beads are binding to protein i increased it to 2% and again it worked perfectly. Now i am doing different tests, i decreased it to 1% to see if it's better, but it didn't change any thing . This is why i am thinking that the probleme is not in the ripa buffer. May be doing the lysis and all the exprience in the cold room will help.
i use a polyclonal antibody, 0.2 ug in 250µl of lysate, and i don't have probleme with the immunoprecipitated protein.
For weak interactions, you can (temporarily) crosslink them to facilitate the precipitation and obtain more reproducible effects. I have used DTBP for this in the past (see for instance: http://www.piercenet.com/product/dtbp). After purification, the crosslink is destroyed and you can load your samples on gel as single proteins.