I've got trouble with non-specific pull down of CENP B in the IgG control IP. Also, really struggling to find a good CENP B antibody for western which probably isn't helping with the IPs...
I've tried changing lysis buffer to 250mM salt (then diluting to run the IP) which has improved things slightly. I have been using TrueBlot beads which has also made a positive difference, but still quite dirty...Does anyone have any tips for this / a good protocol for CENP interactions?
Hi Poppy,
Our lab do not have direct experience with IPing centromeres, but we have IPed many protein complexes including centrosomes. I can't help you with finding a good CENP Ab, but maybe you can utilize an overexpressed tagged CENP B for your experiments if you do not require endogenous CENP B as an alternative.
As for the background issues, usually you can remove background of IPs by increasing both the number and duration of washes with lysis buffer. You can even add RIPA bf as part of your last wash step (some would frown upon using RIPA, but for certain proteins using RIPA bf for making your lysates and for IPs actually helps). Also preconjugating Abs to beads and washing can help reduce background. If you are still having issues, you can crosslink your Abs to beads (such as using dimethyl pimelimidate) although you usually do have to utilize this step unless you are planning to elute your samples instead of direct SDS boiling with 2x sample bf.
Hello, if I'm correct you are working with an essentially nuclear protein, so the first difficulty might be extracting it in a non denaturing condition that you need in order to perform the IP. High salt is one possibility to break the nuclear envelope, that's why 250 mM salt gave you better results than standard isotonic salt. But 250 mM salt will not guarantee a complete break-down of the lamin barrier. You will have to go to at least 300-350 mM salt... although there is no guarantee that at that salt concentration all the interactors of CENP B will still be bound.
Other possibility is to look for buffers offered by many companies for nuclear protein extractions.
Finally, since CENP B should be mainly in the nucleus, you can get rid of the cytoplasmic fraction, this will clean up substantially your IP since 90% of the proteins are cytoplasmic. Just lyse your sample with 150 mM salt and 1% triton or similar detergent and pellet nuclei at 3000 g for 10 min at 4 C. Wash them once in the same buffer and then lyse them in high salt or the proprietary buffers. Use small volumes and dounce briefly in a teflon/glass douncer, then quickly dilute the lysate 2-fold...
Good luck!
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