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Deal all, thank you for all the wonderful helping tips. A quick test of ATP-Mg at 2mM at 37C for 2 hours had proved ineffective in my case so now I'm thinking of trying the other protocols that you had offered as listed below. Many thanks and have a wonderful week! -yong Original Question RE: Removal of bacterial chaperone Hsp70 contaminant from recombinant protein preparation Dear all, I have a protein expressed at 37C for 3 hours in BL21 DE3 and purified with sub-stoichiometric amount of apparent Hsp70 contaminant even after exhaustive affinity (GST-fusion or His-tagged), ion-exchange and sizing column steps. I would like to know if you have a well-established protocol for getting rid of such a contaminant. I asked around and was told to try adding ATP at certain(?) stage of the purification. I would much appreciate your input. Many thanks! –yong @ the Wistar Institute Response 1: Try 1-3M urea in the lysis buffer, and maybe first wash buffer. If you use it in the wash buffer add some lysine (maybe 20mM) to prevent carbamylation of your protein. Kendall Response 2: Hi Yong, I add ATP to my purification. If binding to Nickel column, I would wash with ~10 to20 mM imidazole, then wash with the same wash buffer PLUS 1-2 mM ATP, then rewash the column to get rid of any residual ATP. You can then elute your protein and hopefully be rid of the pesky Hsp70. Good luck, Jill Response 3: I routinely add Mgcl2 (0.5 to 1 mM) in my all buffers and also after purifying protein from Ni-NTA, I incubate it with 1 mM Mgcl2 and 1 mM ATP at 37 C temp for 15-30 minute followed by second step of purification. This way I get rid of HSP contamination.... Hope this will help Radha Response 4: We managed to reduce, but I don't think absolutely completely remove, chaperone contamination by putting ATP in a column wash buffer (while the protein was bound). The idea was that ATP hydrolysis should make the chaperones let go of your protein. The level remaining was only a problem for doing ATPase assays of our protein, which should be nearly inactive in the absence of DNA - even essentially coomassie-invisible levels of chaperone messed up the no-DNA controls. I think my troops also tried adding purposefully denatured goop to the prep to lure away the chaperones, but decided that just complicated things. Phoebe Response 5: I had similar experience once and filling the column with 5 mM Mg-ATP solution and living it like that for few hours, followed by good ATP wash (few more column volumes of Mg-ATP wash), helped a lot. YMMV. Dima Response 6: Have a look at the following papers Proc. Natl. Acad. Sci. (1995), 92, 1948. J. Biol Chem (1984), 259, 8820. Gabriel Response 7: I used Tris 10mM, MgCl2 10mM, ATP 5mM (adjust to pH=8.5, w/o buffer, it would be very difficult to adjust the pH value) to wash the resin (10-20 column volume) during on-column wash, and it works. Yeming Wang, Ph.D. Response 8: Hsp70 is very hydrophobic, and the problem mentioned by you is quite common in many affinity-based purifications. I suggest that you load your sample on an HIC (say, phenyl sepharose) and elute with a gradient from high-salt to no-salt. Hsp70 typically elutes close to no-salt buffers on the HIC chromatography. Shekhar Mande Response 9: If you are trying to purify away a chaperone protein, I found the following protocol useful: Chen et al. (2001) J Mol Biol. 307(1):173-82 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11243812 However, you should also consider the following: If you really can't get rid of HSP70 - even after extensive purification effors - this probably means that the chaperone is binding to your protein (Probably because your protein is not correctly folded, e.g. it may have surface-exposed hydrophobic patches). In this case, even it you manage to "knock off" HSP70 contaminant from your protein, you protein will probably aggregate and/or precipitate. Good luck, Chris Response 10: Perhaps try one of these: 1. Wash with ATP+Mg when bound on the lMAC column (to get it to release your protein). 2. Wash with 0.1% Triton when bound on the IMAC column. 3. Hydrophobic interaction chromatography. 4. Wash with 0.5 GuHCl while bound on the IMAC column (pray!!) On a sad note, the protein will quite often aggregate when it is released. There is a reason why the only sign of target protein you see is stuck in a chaperone... but at least you have the chance to scout around for a good buffer to release it into. Martin Response 11: add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it off during your affinity step...this has worked for me (of course make sure that you're not unlucky and your protein doesn't elute during this step; don't worry it shouldn't) if that doesn't work, maybe try changing some things on the cell growth and expression end, like induction at lower temperature or a different BL21 strain like pLysS or STAR HTH Brad