Immunoprecipitation & Mass Spec analysis - How much mg(ug) of protein lysate is needed for IP followed by Mass Sp (Apr/20/2007 )

Hi folks,

I need to find some binding proteins with my kinase of interest. I used 1mg of mammalian cell lysate stably transfected with

exogenous construct of the kinase for immunoprecipitation, and it was successful to IP my kinase by subsequent western

blotting for that kinase (IP efficiency was almost 100%). Now I want to do large scale IP, run SDS-PAGE gel, stain with colloidal

blue staining reagents and submit the gel to the Mass Spec facility to find some specific binding/interacting proteins with my

kinase. However, I do not think 1mg of cell lystate is enough for staining binding proteins and analyzing them by mass spec

even though it was good for IP-western blotting.

1. At least how much cell lystate do I have to use to IP and subsequently analyze it by mass spectrometry?

2. Also how much protein A&G bead (ul) and IP antibody (ug) do you use for that amount of lysate?

Thank all of you in advance.

---

for recommended protein amount, ask the facility manager as it depends on their equipment; my experience is that ca. 50 pmol should be enough;

---

A good rule of thumb is if you can see your band on a coomassie stain, then there's enough there to work with.

---