I'm looking for suggestions to improve the elution step of immunoprecipitations that doesn't involve reducing agents (such as beta-mercaptoethanol). I've tried 0.1 M glycine pH 3, but the yield is too low compared to what stays bound to the agarose-protein A beads after 3x10-min incubations with that elution buffer. I need to preserve a thiol-based protein modification in these samples.
Hi, this protocol seems reasonable in my hands
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018218
(supplementary data!).
your purpose of eluting protein is for western blot or for some activity assay? because you can elute protein without denaturing agent buy heating them with the native loading dye. I hope you know this, and you need active proteins for downstream applications?
I need to preserve a thiol-based protein modification in these samples. High temperature is known to break it down.
It is tough! I tried also similar things with from HA antibody immboilized on agarose beads, but no use. Company can promise anything but in reality it doesnt work for me too(elution under low pH).
I am curious to know form Steingrimur about elution with ethanol!
Hi Ananda. I was using protein A sepharose to IP proteins from serum. I needed a solution that knocked the protein off the resin and was easy to concentrate afterwards. I tried some low/high pH buffers, but they didn't work for me as well as 75% ethanol.
I am not sure what antibody you are using and some of these methods involve extreme pH which might denature your protein but here is a list of reagents that might elute your protein. These pH elutions might also require neutralization to prevent precipitation.
- Glycine 0.1-0.3M pH 2-3 (increasing concentration or lowering pH further might be useful)
- Citric acid 0.1-0.2M pH 3-4
These next two are well suited for membrane proteins
- Glycine-NaOH 0.1M pH 10.5
- Sodium borate pH 10
You can also try neutral pH with some commercial reagents. pH elutions are known to give lower yields so I am not sure if any of them will help you. You might have to prepare a larger sample or make a protein with a tag and elute it by cleaving.
If competition doesn't work you could clone your gene in a TEV-site (or whatever cleavage site suits your needs) vector and simply cut off your POI from the tag after IP with the corresponding protease. Works even better if the protease has a tag itself like PreScission protease from GE which has a GST-tag. This allows you to remove tag and protease from your POI in one step.
if the elution efficiency is too low, maybe the pH in the reaction tube is not low enough. Before you elute you have to make extra sure you have taken off ALL of the remaining IP/Wash buffer. Any remaining buffer will dilute your glycine buffer and the pH shift will be to small.
Hi Luiz, I usually use magnetic beads for IP to apply in proteomics approaches by MALDI-TOF and the elution is carries out with 0.1 %-0.5 % TFA aqueous. Or formic acid for IT MS
Hi, as mentioned above, try yet lower pH (below 3) or chaotropes (6M guanidine HCl or 8M urea). Commercial buffers usually do not work in these "special" cases. What do you want to do with the protein? do some analysis (MS?) or activity testing?
Hi, this protocol seems reasonable in my hands
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018218
(supplementary data!).
if you only plan too test your sample by western blotting, then don´t elute w acid, but add the sample buffer- that you would add to your sample to perform SDS PAGE- as a elution buffer for example , boil then the whole resin, spin, discard agarose, keep supernatant and run as it is on your gel. But still, this method would not allow to make any assay where you would test the functionnality of your protein. I hope this helped
Hi Luiz.
If your target protein is thiol containing one, you can purify it by covalent chromatography using 2'-pyridyl immobilized beads. This method allow you to capture and detach the protein very easily from the beads by DTT elution.
I agree with the Christoph Ufer comment's about the Elution precedure. You need to exchange the whole washing buffer with the elution buffer to get the good yield of purification.
I carried out non-reducing IPs in this paper:
http://www.ncbi.nlm.nih.gov/pubmed/20807509
It's important not to add reducing agent for SDS-PAGE. pH should be roughly neutral if I remember rightly.
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