The protocols for (brain) tissue digestion I am trying out are: lysis (buffer containing 0.1% SDC or SDS in 100mM TEAB + phosphatase inhibitor which is sort of yellow), reduction with TCEP, alkylation with iodoacetamide, digestion with trypsin, and acidification with trifluoroacetic acid (TFA) to deactivate trypsin. After that I usually dry the sample and do a C18 clean-up.
For some reason my samples become yellow after the addition of TFA (and this was also the case with lysis buffers with SDS or SDC so I don't think it is detergent related). As this happened to a colleague who didn't use the phosphatase inhibitor, I don't think it's related to it either. When I do the C18 clean-up this yellow goes to the eluate (elutes with peptides). Would anyone know why this yellow colour occurs? The most disturbing thing for me is that it does not always happen to every sample, as you can see in the photo 0.1%SDC is clear.
Moreover, for the last digestion, I decided to try out Rapigest (0.1% in 50mM TEAB). When I added the TFA for acidification step, the sample was still clear, but after a while drying it in SpeedVac, when I checked up on it, it had a bloody red pellet-like oddity as you can see in the photo. I left it in the SpeedVac to dry out completely, and when I checked it again, the red colour disappeared and it was clear-whitish again. Would anyone know why this happens and if it interferes with subsequent steps such as C18 clean-up or LC-MS identification? So far, I have asked my colleagues, but they have never seen it before and are calling it magic.