Changing dialysis buffer every half hour and then dialysis ON - protein precipitation prevention (Aug/19/2009 )
Hello ya'll!
I read on a forum similar to this one that changing the dialysis buffer several times before eventually leaving it ON helps prevent protein precipitation. I dont understand why this would be true, surely that dilutes my protein solutions even more and might promote precipitation?
What other factors could I take into account for succesulf dialysis? Im supposed to dialysis into MOPS and 100mM NaCl pH 7.9 after eluting in 100mM NaCl lysis buffer pH 8 + 300mM imidazole (pH 9). Washing occurs with 300mM salt buffer.
I would really appreciate some help, I came to lab to day to find a funny little cloud of protein lying around. I have decided to start all over again and would love some tips!
The kinetics involved in dialysis are somewhat complex and difficult to control. Surface/volume ratio is a factor, so are total volumes of dialysate and buffer. The most important factor though is the buffer that you want to dialyze into. If that buffer is not compatible with your protein, i.e. crashes out your protein then there's no way you can 'trick out' the system.
The way to go would be to first set up lots of small experiments where you check which buffers do and which ones don't precipitate your protein. There's a free trial going on with a protein solubility screening kit - OptiSol from Dilyx Biotechnologies (www.dilyx.com) - that you may want to try out to determine which buffers work and which ones you may better avoid. Short of setting up 96 dialysis experiments, you could do second best and just dilute with the supplied solutions, run the assay and identify the buffer system to dialyze into.
My 2 ct,
DaveD
PhD on Aug 19 2009, 12:04 AM said:
I read on a forum similar to this one that changing the dialysis buffer several times before eventually leaving it ON helps prevent protein precipitation. I dont understand why this would be true, surely that dilutes my protein solutions even more and might promote precipitation?
What other factors could I take into account for succesulf dialysis? Im supposed to dialysis into MOPS and 100mM NaCl pH 7.9 after eluting in 100mM NaCl lysis buffer pH 8 + 300mM imidazole (pH 9). Washing occurs with 300mM salt buffer.
I would really appreciate some help, I came to lab to day to find a funny little cloud of protein lying around. I have decided to start all over again and would love some tips!