Reduction, alkylation and digestion of protein in solution

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

Samlple : BSA (10 pmol)

Enzyme : Trypsin (1 pmol)

Chromatography : C18 reversed phase HPLC

1. [Reduction] Incubate a protein sample in the presence of DTT at 60 °C for 30 min.

		(final conc.)
10-100 pmol/µl protein	1 μl	(10 - 100 pmol/10 µl)
0.1 M Tris-HCl, pH 8.8	2 μl	(20 mM)
0.1 M DTT	1 μl	(10 mM)
0.1% SDS	1 µl	(0.01%)
Acetonitrile	1 μl	(10%)
DW	4 μl

---

Total

10 μl



2. [Alkylation] Add acrylamide solution and incubate for 30 min. at rt.

		(final conc.)
+ 0.5 M Acrylamide	1 μl	(50 mM)

---

Total

11 μl




(*) Iodoacetic acid, Inodoacetamide, or 4-vinylpyridine can be used instead of acrylamide. To make 100 mM 4-vinylpyridine, dilute neat 4-vinylpyridine 90-fold with water.



3. [Digestion] Add enzyme solution and incubate overnight at 37°C.

   		(final conc.)
   + 1 pmol/µl trypsin or Lys-C	1 μl	(1 pmol/20 µl)
   + 10% Acetonitrile	8 μl	(10%)

   ---

   Total

   20 μl

4. Recover the enzyme solution.
5. For protein sequencing, apply the enzyme solution directly on an HPLC with a C18 reversed phase column. For peptide mass fingerprinting, desalt the recovered solution with a Ziptip µC18, and apply on a MALDI target plate.