Once again, thank you for your replies
Problem is I need my protein material soluble DURING proteolysis as well not just before loading it on the column. Glycerol proved to be the only reagent that kept solution clear without compromising my proteaseís stability. Sucrose could be another alternative but I doubt it could prevent bubbles forming due to its high viscosity. DMSO (up to 10%) didnít work.
We donít have a degasser so my colleague and I were experimenting blowing helium through our buffers using available material, which didnít help. The only protocol that worked well included a very steep linear gradient, 10 min at 1 ml/min 0-100% ACN, apparently not allowing enough time for bubbles to form but the peaks came out too stacked of course. Now that really became a problem when I started running BME-reduced samples so I finally opted for a step gradient. Thereís been no bubbles with the step protocol so far (and Iím groping for wood
) so right now Iím trying different protocols to separate both BME and urea from my reduced fragments, which is a bit tricky.
P.S. I wonder why I canít view all of the replies to my post, only the last two. There were a lot more, were they deleted?