I am preparing samples for phosphoproteomics. After reduction, alkylating, and digestion, I acidified my cell lysates with 20% TFA before purifying the digest with Sep-pak. When I digested ~1.5mg protein, I got precipitate in my acidified digest, but when I digested ~0.7mg protein, I got a little milky solution with no precipitate after centrifuge. Can I use the milky solution? Where did the precipitate and milky solution come from?
After I centrifuged the acidified digest with precipitate and got the clear supernatant, and purified them with Sep-Pak. Then I dried my peptides with Speedvac. As a result, I got some yellowish pellet. I thought the pellet was supposed to be white or invisible, and the Sep-Pak can eliminate almost all the background. Where did the yellowish pellet come from?
Thank you very much!
Ran：As a young scientist, you may committed a common mistake by overly using too much amount of proteins. For any proteomics study, 100 micrograms of proteins are enough for analysis, along with proportional amount of trypsin or chymotrypsin. The solutions should be clear and no visible color shall be seen. Good luck.
Ran：If the precipitate or milky color persist after reducing the protein amount to 100 microgram level (which is unlikely), then its possible that your trypsin solution may contain calcium ion and form precipitates with phosphate in the sample buffer.
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