Protease digeston on PVDF membrane

Laboratory for Biomolecular Analysis, Institute for Protein Research, Osaka University

Samlple: BSA (20 pmol) --> SDS-PAGE --> PVDF membrane (Immobilone PSQ).

Enzyme:Trypsin (1 pmol) in 30 µl of 20 mM Tris-HCl (pH 8.8), 10% acetonitrile.

Chromatography: C18 reversed phase HPLC

After SDS-PAGE, electroblotting, CBB-staining, and cysteine alkylation, wash the membrane with 10% acetonitrile. If the membrane was dry, soak it in methanol first.

Incubate the membrane in 0.5 ml of 0.5% PVP-40 /100 mM acetic acid for 30 min at rt. on a shaker or rotator.

Wash the membrane with 10 % acetonitrile (5 min x 3 times ).

Incubate the membrane in 30 µl of enzyme solution (*1) overnight at 37 °C.

		(Final conc.)
1 M Tris-HCl, pH 8.0 - 9.0 (*2)	20 µl	(20 mM)
Acetonitrile	100 µl	(10%)
10 pmol/ml trypsin (*3)(*4)	3 µl	(1 pmol/30 µl)
Water	877 µl

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Total

1 ml


(*1) If the membrane was not covered with 30 µl solution, add more enzyme solution.
(*2) Ammonium bicarbonate can be substituted for Tris-HCl.
(*3) Use sequence/proteomics grade trypsin (e.g. Promega, #V5111). Avoid repeated freezing and thawing.
(*4) Lys-C (e.g. Wako, #121-05063) can be used instead of trypsin in this protocol.


Recover the enzyme solution to a new tube.

Rince the membrane with 30 µl of 10% acetonitrile/0.1% TFA and combine it with the solution obtained in Step 3.

Inject the sample into a C18 reversed phase column on an HPLC system.