My protein extraction methodology from bacteria produces a light brown pellet. I have been toldthat these colours or pigmens can be problematic for the downstram mass-spec analysis on the Q executive.
My extraction conditions are cell wash buffer, cell extraction buffer which includes phosphatase inhibitors (including sodium vanadate, Na fluoride, Na pyrophosphate ), heat step, sonication, DTT, PMSF, benzonase and N-octyl glucoside. The proteins are precipitated using acetone/ethanol/water 4:4:1 overnight resulting in partly dark green pellet which becomes browner upon air-drying. The supernatant from the total cell extract looks straw yellow. May be some type of reaction between the sodium vanadate/Na fluoride and the DTT but I am not sure!. My next step is dialysis of the sample in urea buffer. Any helpful explanations gratefully appreciated.
Why does the color concern you? I've seen a lot of coli lysates, from both alkaline and sonication, and they range from yellow to really dark brown.
Hi Adam
I was informed that for the downstream analysis of the trypsinised enriched peptides, that the mass spectrometry analysts preferred not to have pigmented samples. We are analysing phosphopeptides.
I see. Do you know anything else about your protein? Chromatographically separated proteins are almost always a more pure sample.
Well it could be from your media, media components often co-purify. Many proteins are brown or dark in color.
Ithink I have part of the answer ! In our homemade phosphatase cocktail there was no colour development, whilst in the mixture we purchased from a company , a brownish colour started to develop immediately on addition of DTT. The only difference in both cocktails are sodium-molybdate, sodium-tartarate and imidazole. I suspect the colours I see in the protein pellet is a result of reduced forms of molybdenum. But there is still some pigment left from the growth of the bacteria in late stages which we are working on.
Bacteria may contain brown pigments precipitated or Some phenolic compounds gets brown when oxidised.
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