In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresis (According to EMBL Protein and Peptide Group, 1997)

MB 11.12.00

1. Excision of protein bands (spots) from polyacrylamide gels

Rinse the gloves you use with water to avoid traces of dust in your sample.

2. Reduction and alkylation

This step is omitted because proteins are already reduced and alkylated during the equilibration after the 1st dimension. Include maybe for small protein amounts.

3. Washing of gel pieces (Coomassie, Sypro Ruby)

Washing to remove unpolymerized acrylamide (which may react with the peptides) and other (buffer) contaminants (which may give a higher background during MS measurement)

Repeat this washing step twice.

4. In-Gel digestion with Trypsin

5. Extraction of peptides from the gel piece

At this point, samples can be stored at -20°C.

6. Mass spectrometry

2. Reduction and alkylation


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