I'm currently trying to analyse the milk and fermented whey's protein profile using SDS-PAGE. I've already tried with Tris-Glycine and Tris-Tricine, but I always keep getting a smeared and poor resolution gel. The samples, after denaturation with sample buffer (SBM) at 100 ºC for 10 minutes, were centrifuged for 10 minutes and some of them show a white film on the surface (possibly lipids, because I have already tried some delipidification treatments and it disappeared) and also a white pellet which does not seem to dissolve. I'm currently struggling with getting a good gel, so if anyone could help it would be great!
Measure your proteins before loading--never more than 25 ug in these cases--don't boil too much---2 minutes at 100 deg is enough to denature
How prevalent is Asp-Pro cleavage in Laemmli buffer? I've worked over the years with several proteins containing DP motifs and I'm yet to witness this phenomenon. Either it is sequence context-dependent or depends acutely on temperature (I don't boil my samples, I use 95*C).
Your delipification step is good. Coagulation of protein with acetone or ammonium sulphate and centrifugation will give a good amount /concentration of protein. Alternatively you can coagulate your protein with TCA. Then regular Laemlli procedures will give you the protein bands. If the majority of your proteins have low molecular weight due to the fermentation process, you might increase the % of acrylamide gel and ajust the voltage
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