Lost: protein. Last seen prior to purification. - not present in flow through, washes or elutions! (Aug/22/2006 )

Hi,

I read the other thread on this problem, but my loss appears to be happening during centrifugation (or earlier) rather than being a problem with binding to the beads in the column, because I could not detect my protein in the flow through, washes or elutions

My protocol is as follows (after running sample from induced cells to confirm expression of recombinant protein):

Resuspend cell pellet in 50mM Tris-HCl pH 8, 10mM beta-mercaptoethanol, 0.02mg/ml PMSF, 0.03mg/ml benzamidine.

Add lysozyme to 0.2mg/ml final concentration, stir in cold room for 30 mins.

Add MgCl2 to 5mM final concentration followed by DNaseI to concn 0.01mg/ml, stirred again for 30 mins at 4 degrees.

Add cholate (detergent solution = 50mM NaHEPES pH 8, 3mM MgCl2, 50mM NaCl, 200g/L cholic acid) to final concn 1% (w/v). Stir again for one hour, 4 degrees.

Centrifuge at 30000rpm for 40 mins.

Then, i mixed the supernatant with beads and proceed with purification, but my protein is nowhere to be seen.

I am assuming that this is because my protein has pelleted with the cell debris during centrifugation? I thought that the use of a detergent was supposed to stop this from happening.

I thought that perhaps I was stirring the proteins too viciously and mashing them up? Is that possible? Other proteins can be slightly seen in the elutions, like the usual background I get when I purify other proteins.


Please help me I don't know how to fix it!!

check everything by SDS-PAGE.... also the pellet can be redesolved in SDS-samplebuffer for analysis. Also cook a fraction of the beads with sds-buffer. maybe the protein just isn't eluted?!
have you checked that your protein is expressed?
the detergent will not always prevent the precipitation of a protein....

Yeah, save a little bid of your sample from each step and run a gel on all of them then you will know what is going on, easily.