Dear all, I am working with a multi-subunit complex which needs a minimum of 5% glycerol for stability in solution. I usually reconstitute this complex together with multiple interaction factors as a part of big ensemble over GraFix for cryoEM grid preparation in a final buffer containing 1% glycerol. The initial rationale was that in the presence of the interaction partners, my relevant multi-subunit complex will be happy in a low glycerol environment. Turns out, it falls apart from the rest of the complex in the course of the buffer exchange from 5% to 1% glycerol. I know this to be the case, because I screened independently for the multi-subunit complex alone in buffers with 5%, 2.5% and 1% glycerol respectively and could only see and reconstruct particles from preparations with 5% glycerol. I have tried supplementing the 1% glycerol buffers independently with 0.005% NP40, 0.005%DDM and 0.004% OG to no avail. As a next step, I would like to try amphipols and hence seek expert opinions on the following questions: 1. For the structures determined with the amphipol A8-35, the protein was incubated with an excess of A8-35 and removed by SEC. Now, if i pre-purify my multi-subunit complex with A8-35 or its likes, will it inhibit the higher order complex formation with my other factors? 2. If inhibitory, I could also add the amphipols after my GraFix experiment before exchanging into the 1% glyecrol buffer. In that case, would I be able to remove the excess amphipols with dialysis?
I am also happy to receive other suggestions apart from the use of amphipols to stablise the complex. Many thanks in advance, Srinivasan ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
