Hi all,
This is my first post here and I am now thinking to myself why I haven't tried this before! Anyway, I'm wondering if anyone could lend some of their expertise to help me figure out how to successfully perform a CoIP. I've been working on it for some time now and have seen and tried many different protocols so I definitely know the basics; although truth be told, I am fairly new to biochemical research in general.
I'll try to keep this brief for now but specifically I'm trying to perform a CoIP by precipitating eNOS and detecting it's supposed interacting protein partner Hsp90 (with Western blotting). I'm using dissected sections of rat aorta, and I feel that this is where some of the issue lies. Essentially all of the protocols I've found use cell culture samples and not whole tissue. I am not too certain how to properly homogenize/prepare this sort of sample in order to preserve the eNOS/Hsp90 interaction. Should I be using glass/glass homogenizer or is an electric homogenizer fine? Should I be using mild detergent in my buffer or not? What sort of salt concentration would be recommended? Should I be homogenizing the tissue (and even performing the CoIP) immediately after dissection or after freezing? (I have tried altering some of these things without much luck so far).
As for the CoIP itself: because the aorta is so small, I have a limited amount of protein to work with. I have had some success precipitating eNOS (without Hsp90) using only 100 ug total protein (concentration: 0.67 ug/uL) so I've been sticking with this for the CoIP. I use an antibody already conjugated to agarose so I skip the Protein A/G step. I haven't been blocking the bead/antibody but I'm not really getting a lot of background on my Westerns so I'm not sure how necessary it is. I wash the beads 3 times with TBS (25 mM Tris, 150 mM, pH 7.2; I've tried homogenizing with this solution also). Finally I elute the proteins using SDS-PAGE sample loading buffer (Laemmli buffer) with heating.
There are a lot of other subtle details of course but I thought leave it at that for now. At this point, I'm not too sure what to do next so if anybody has any experience with this and can provide suggestions, I would greatly appreciate it. Thanks.
OscarC
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