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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

GTP Blots for the Identification of GTP-Binding Proteins

GTP Blots for the Identification of GTP-Binding Proteins
Contributor: The Laboratory of David Drubin at the University of California, Berkeley
 
Procedure
1. Electrophorese the protein sample on a polyacrylamide gel (see Protocol on SDS-PAGE) and transfer to nitrocellulose (see Protocol on Western Blotting).

2. Rinse the blot with Binding Buffer.

3. Soak the blot in Binding Buffer for 4 hr to overnight at room temperature.

4. Incubate the blot in Binding Buffer containing α-[32P]-GTP at 1 μCi/ml at room temperature for 60-90 min.

5. Rinse the blot several times with Binding Buffer and incubate the blot in Binding Buffer while shaking for 1-2 hr, changing the Binding Buffer every 15-20 min.

6. Air dry the blot and expose it to film (see Protocol on Autoradiography)

Solutions
Binding Buffer   0.1% (w/v) Bovine Serum Albumin
30 μM ATP
5 mM MgCl2
0.3% (v/v) Tween-20
1 mM EGTA
50 mM Tris, pH 7.5
 
BioReagents and Chemicals
ATP
Tween-20
Magnesium Chloride
Tris
Bovine Serum Albumin (BSA)
Iń-[32P]-GTP
EGTA
 
Protocol Hints
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