Service offered

In gel tryptic digestion

Preparation of Gel Pieces containing Protein Spots of Digestion

1. Excise the gel pieces containing the protein spots of interest. (Be careful to cut only the gel piece with the protein spots, avoid excess gel outside the protein spots, that could interfere with the analysis to be carried out and also could contain other proteins that are not visible).
2. Wash the gel pieces twice in 50% acetonitrile
3. Wash the gel pieces twice in 50% acetonitrile containing 50 mM ammonium bicarbonate, pH 8.0.
4. Wash gels in 50% Acetonitrile containing 10 mM Ammonium bicarbonate.
5. Gel pieces are finally dried and used for tryptic digestion.
6. Trypsin is dissolved in 25 mM ammonium bicarbonate, pH 8, to a final concentration of 1 µg/ml (the concentration of trypsin could be increased depending on the size of the gel pieces). For now this is a good guideline. To each tube add 50 µl trypsin and allow the gel to absorb the added solution.
7. Add enough buffer to cover the gel and incubate overnight at 37°C.
8. Solution containing some of the peptides that are released into the buffer are collected.
9. Gel pieces are extracted twice with 0.1% TFA in water for 20 min and the soluble fraction is pooled with the corresponding fraction above.
10. The gel pieces are then extracted twice with 60% acetonitrile in 0.1% TFA. Corresponding fractions are pooled together and dried. The final pellet contains most of the tryptic peptides from the digest and is ready for analysis by MALDI-TOF.

Data Provided

Picture of gel, extracted and lyophilized peptides.

Turn-around time

5 working days

Fee schedual

	MCW	Others
protein digests	$150	$300