co-IP trouble shooting - (Oct/19/2009 )

Hi, all,

Greetings to everyone. My first post here. I am happy to find a nice place to communicate with others experiences and problems in biology research.

I am doing a co-IP using Sigma anti-flag M2-resin to IP my bait protein (Flag-tagged, ~60KD), and after washing the resin beads, I boiled with 2xloading buffer at 100℃ for 10min.
The elution was then subject to Western detection for my prey protein (Myc-tagged, ~110KD), using anti-Myc 9E10 antibody.

Both of the two protein are expressed well as suggested in the input lanes from western. For the co-IP lanes, however, there is a clear band around 120KD (a little bit above the prey
protein band shown in the input lanes). There is no such a 120KD band in the inputs. I am not sure if this 120KD band is actually the co-immunoprecipitated prey protein. I know it
might be verified by using the antibody against the prey protein itself, but such commercially available antibody is not working good. Another way is to do the co-IP in the control sample
which is transfected with either bait or prey protein, to see whether a similar 120KD band will show up. This is what I will do next.

Anyway, my question is is it possible that the prey protein from the co-IP sample might show up at a slightly different position compared with the prey protein from the inputs?
I mean since I use 2xloading buffer for the co-IP sample while 1xloading buffer for the inputs, will this different concentration of sample buffer cause different migration speed?
If I want to achieve 1x for the co-IP, I need to first elute the co-IP samples from beads and then add loading buffer to make a final of 1x concentration. What would be the efficient
elution method for this purpose? Use 3xflag peptide(150ng/ul) @4℃, 30min for competeion elution? Elution with 0.1M glycine at pH 3.5 for 5min? Or can I just add some lysis buffer
to the beads and boil at 100℃ for 10min? I am not sure which one has highest efficiency.

Did you ever encounter similar situation? Any comments and suggestions are appreciated. Thank you very much.

Hi there,

sometimes in Ips I saw changes in the size of the protein immunoprecipitated...dont ask me why!!! But it could be. Anyway you can use some controls. So, if you are doing a Ip with flag and then wb with Myc you should have a sample without flag, and sample with flag and myc. Another control you can use is IgG: you split your samples in two, and then you Ip one part with flag and the other with IgG.

For the elution part:

You can use sds 1x to do the elution. Normally I do it with SDS buffer 1.5X and boil 5 min (you add your buffer to the dry beads).
You can use as well flag peptide, incubate with it for 1 hour and then take the supernatant add SDS (final concentration 1X) and boil