after purification/storage/buffers... - How not to lose protein after purification (Mar/23/2010 )
I have my protein in Phosphate buffer (pH 7.4). During purification the phosphate buffer contained NaCl, NaN3, and EDTA. Now, I am doing dialysis and using just phosphate buffer with NaCl . I am afraid that now having the protein without NaN3 preservative, it will be eaten by bacteria (or just degrade) . Also, I am not sure what the role EDTA played in the buffer, why not to add it now? However, I think to sterilize the protein and then I will do protein conjugation with fluorescent labels and PEG - may be it is even for best I am not using EDTA and NaN3? What do you think?
First of all, doing your work in the cold will prolong the life of your protein.
Next, EDTA is there to chelate any divalent cations, reducing the risk of protease digestion. As for the azide, it probably isn't necessary, as the dialysis tubing should be sterile, and bugs really can't get in, but again, it can't hurt.
Looks like a straightforward experiment to me: just give it a try. Snap freeze a small sample, go through your procedure and then compare on SDS-PAGE. See any degradation?
You could always add the Roche protease inhibitor tablets. These are expensive though.
Or add PMSF like in the old days.
As a general rule, working quickly and with samples chilled 'on ice' may be all you need.
my 2 ct
ysia2000 on Mar 23 2010, 04:25 PM said:
NaCl is for ionic strength for correct folding. EDTA to inhibit metalloproteases; dialyse at 4°C, so you do not have to work with preservatives;
sterilizing can be performed by sterile filtration, however, you may lost some protein by unspecific binding to filter and plastic of syringe; so do not filter if your amount of protein is limited