In-gel trypsin digestion

[crd]

Does anyone have a good, reliable in-gel trypsin procedue, with good recovery from the gel subsequently.

Thanks

[proteaPam]

Hey crd,

The following is a good reliable [Only registered users see links. ]


As an alternate for extraction protocol after overnight digestion I have centrifuged and collected the supernatant in a clean tube. Dehydrated the gel pieces with 50 μL 100% Acetonitrile and incubate at RT for 20 mins. Pooled together both supernatants in the same tube and dried down with Lyophilizer or SpeedVac. Once dry, I typically recommended an acetic acid clean-up (50 μL of 0.1M Glacial Acetic Acid). Dry down in lyophilizer or SpeedVac.

If this is for mass spec. analysis I would recommend clean-up of the salts with [Only registered users see links. ] or [Only registered users see links. ].

Good luck!

Pamela Williams
Laboratory Technician at Protea Biosciences
[Only registered users see links. ]

[sharedproteomics]

Yup. That is a pretty standard in gel digest procedure. I like SepPak columns from waters for cleanup but it they might be too much for lowly abundant proteins in a gel band.