Co-IP experiments, Ratio of amount of cells/lysis volume/ug antibodies - (Apr/27/2006 )
I want to show protein-protein interaction by co-IP, using not-transfected cells in which my proteins of interest were very poorly expressed or at least with "normal" level.
So, I already known that I have to test alternative lysis buffer, antibodies concentration and to fix electrophoresis and transfer blotting conditions.
Moreover, because my limiting factors are the slow growing of my cells and the expensive price and limited stock of my antibobies (!!), I'd like to got some advices about the number of cells or the volume of cell lysate that I can use for co-IP in 1.5ml epptube to expect detectable signal after ECL western blot ?!?....
Some protocols for normal western blot use until 1 million cells for 50 ul volume loadind/well (on mini gel system) and for co-IP, until 5 million of cell for one co-IP (1.5 ml epp tube) load in one well (40ul) after elution. Could someone tell me if these proportion were possible and in these conditions which is the amount of antibodies (<ug>) recommand to immunoprecipitate protein amount extract from <5> million of cell ?
I think use "big" gel electrophoresis system and then a possible loading volume of 100ul/well after elution of protein from dynaBeads-Ig complex...
I will be very happy to got some advice from other people working with not-transfected cells and not overexpressed or recombinant proteins cell models... If you could give me some examples of your ratio number of cells/lysis volume and ratio of number of cell/ug of antibodies coated on beads for co-IP, etc.....
I thank you in advance...
P/S : Please, excuse me for my so really bad english...
since u r restricted in terms of ur protein of interest and antibody concentration, i would prefer playing around with time of incubation.
increase the incubation time with what ever amounts of your protein of interest and antibodies available.
it is little bit tough to say which is the ideal situation in ur context,
in general 1-10 ng of protein can be detected reproducibly.
if you know little bit about ur protein expression levels in cells, based on this u can change the concentration of antibody and find the optimal condition.
more over, we dont know how specific and efficient your antibodies are?
so standardisation is the only way u can get suitable answer for ur situation.
I've been working with a very low expressing protein using a retroviral construc with a very weak promoter. I have been most successful using as many cells as possible - i.e. 4-5 million in a 150mm plate using 1mL of lysis buffer. Again try to load as much protein as possible into the IP and incubate overnight. I have gotten signal w/ 700mcg but mor is better. As to the concentration of the antibodies - it's pretty much a crap shoot I'm afraid.