Posted 15 October 2010 - 01:46 PM
Thanks for your help.
Posted 17 October 2010 - 06:00 AM
Do you know whether the soluble form of the protein is 'active' / in the 'open confirmation' for the interaction to occur ?
I have read that there are some proteins which needs to be activated before it can open its confirmation to allow other protein interactions. If that is what happens in your case, I guess it is possible that the interaction happens only when the 'active' protein translocates to the membrane.
What do you think ?
Posted 21 October 2010 - 06:19 AM
Co-IP usually refers to immunoprecipitation of a complex of different proteins using an antibody towards one of the proteins in the complex. I am assuming that the antibody you are using does not target the protein in question directly? In which case, if this is a membrane-associated complex, the complex will in fact only occur in the membrane fraction. The protein in question may well be in the soluble fraction also, but here it is not associated with the other proteins in the complex and hence is not captured using your antibody.
Another feasible explanation is the stability of the protein complex: Some protein complexes are highly unstable and will dissociate quickly. Invariably, the stability of a protein complex also depends on the conditions it is in, e.g. salt concentration, detergent type and concentration, pH, temperature etc. As you have different fractions, I assume you are using different lysis conditions for the different fractions? It may well be that your complex is more stable in the conditions used in the membrane fraction than in the soluble fraction.
Time is also a determining factor in co-IP. The time of dissociation varies greatly between different complexes. In addition, when doing IP, non-specific binding will tend to increase linearly over time, so it is advantageous to isolate the complexes quickly. Isolation should be done immediately after lysis (and at the same time from different fractions!). It is an advantage if the capture matrix used for the co-IP has fast reaction kinetics, as this will improve the likelihood of complexes not having dissociated and also give higher signal:noise ratio. Cristea et al. has done a great study on optimization of co-IP: http://www.mcponline...cprot;4/12/1933
We recently launched a co-IP kit which makes it all very easy of course. Read more here if you are interested:
Hope you find this helpful. Good luck with your isolations :-)