Reduction and alkylation of protein cysteine residues on PVDF membrane

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

Protocol

1. After SDS-PAGE, electroblotting and CBB-staining, excise protein bands from the PVDF membrane.
2. If the membrane is dry, submerge it in methanol for >20 sec.
3. Rinse in 10% acetonitrile.
4. [Reduction] Incubate the membrane in reducing solution (10 mM DTT/10% acetonitrile/100 mM Tris-HCl, pH 8.8) at 60 °C for 30 min.

		(final conc.)
0.1 M DTT	10 μl	(10 mM)
1M Tris-HCl, pH 8.8	10 μl	(100 mM)
Acetonitrile	10 μl	(10%)
DW	70 μl

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Total

100 μl

5. Remove the reducing solution.
6. [Alkylation] Incubate the membrane with an alkylating reagent such as acrylamide, 4-vinylpyridine (4-VP), iodoacetic acid, or iodoacetamide for 30 min at room temp. For protein sequencing on ABI Procise protein sequencer, 4-VP is often used since the PTH-amino acid standard mixture for Procise contains pyridylethyl cystein (PE-cys), the derivative of 4-VP.
   
Alkylating solution:

		(final conc.)
100 mM acrylamide (*)	10 μl	(10 mM)
1M Tris-HCl, pH 8.8	10 μl	(100 mM)
Acetonitrile	10 μl	(10%)
DW	70 μl

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Total

100 μl




(*) Iodoacetic acid, Inodoacetamide, or 4-vinylpyridine (**) can be used instead of acrylamide.

(**) 100 mM 4-vinylpyridine can be prepared as follows.


4-vinylpyridine	1 μl
DW	92 ul

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Total

93 μl



7. Rince 3 times with 0.5 ml of 10% acetonitrile.
8. The membrane is now ready for N-terminal sequencing or enzyme digestion.

Notes

1) For mass spectrometric analysis of gel-separated proteins, acrylamide is recomended as the alkylating reagent because proteins separated by a polyacrylamide gel are often modified by free acrylamide in the gel. If one of other alkylating reagents was used for post-electrophoretic reaction, mass peaks for cys-containing peptides could split, resulting in decreased score for database search.