Hi all,
I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by performing extraction under denaturating conditions using an urea buffer. (As it might be that the biotag on the relatively big LDB1 complex is not exposed enough to bind to the streptavidin beads).
I first precipitate the nuclear extracts with TCA, then i pellet the extracts and add urea buffer to the precipitated extracts. I've tried urea buffers of 4M, 5M, 6M and 8M with 2% SDS 0,2M NaCl and 100mM Tris. The protein pellet did not dissolve properly in these buffer, even though I had such a high concentration of SDS. I still added the beads and left them rotating overnight at 4*C. The 6M and 8M buffer formed a weird gel like precipitate, which i know is because of the low temperature. But I can't imagine doing a overnight protein+bead incubation at room temp. I ran a western blot and there clearly was bioLDB1 visible, but a lot less compared to a non-denaturating extraction method. So my question is: how can I optimize my urea buffer (should i add more NaCl to dissolve the pellet) or does denaturation not optimize protein extraction at all?
Maybe suggest the use of this method (do you want the protein, or do you want to conserve It's properties? ) urea, SDS, 2Me (denaturing, chaotropic agents, maybe fI) are all important factors to bare in mind. (Denaturing only helps to a certain point) maybe suggest the chains are not properly folded).
13.6 pKa of urea buffer and is mw is 60 roughly. Do you think the protein might need some stabilizing agents, what I don't Know ir it may be to many. Hoffman series might help. Gn,,... Ca, cl-.
I'm not sure if you need to try biotin, strapetA (specifically). Maybe the core of the protein is too packed, hydrophobic and you might need unload it. SDS does damage, you did not see any change in the concentration? Should those beads have some kind of binding? Maybe try the NaCl, (not always does the denaturing agents help). I would like to know if you could add a step in the process, where you add the components and then go on.
Mª Angeles Zorrilla Lopez-Perea : It's Hofmeister (doi:10.1007/BF01838161)
TCA precipitation leads to protein pellets that re-dissolve poorly. I'd either do the precipitation in the presence of desoxycholate as carrier (doi: 10.1016/S0003-2697(76)80064-4), or use methanol/chloroform (doi:10.1016/0003-2697(84)90782-6) instead.
I agree on the TCA. I must be careful with Hoffmeister. I do hope it was helpful. Maybe say the add on is desoxycholate. What I don't know if changing the beads could do. (Agarose) . Western blot does help.
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