My thesis involves the analysis of differential expressed proteins in Listeria monocytogenes when exposed to sublethal concentrations of nisin, a well known bacteriocin used in the food industry. I've tried to make 2DE gels but unfortunately I couldn't make good gels in order to compare the control and treatment situations. Now, we established a gel-free approach and were suggested to use a MudPIT analysis using spin columns (SCX column followed by a C18 column) for further Mass Spectrometry analysis. The question is: How is it done? Since i don't have much experience in this kind of methodology, I wonder if there is a certain guideline for it (buffers, how to prepare the columns...)

Another question is regarding the salt step. What is the optimal concentration of salt needed to make the peptides come out slowly, avoiding the 1st elution tube being highly concentrated and the last ones with almost no sample eluted?