Team:Brown/Notebook Protocols/immunoblotting

''' Immunoblotting (Western Blotting)

Adapted for Spot-Blotting '''

Reagents needed: 1) Blotto (per liter, 10.8 gms NaCl; 20 mM Trip, pH 8.0; 30 gms not-fat dry milk; 0.01% Tween-20)

2) Antibody (anti-6-his, in blotto, diluted 1/10,000)

3) Alkaline phosphatase buffer, (per liter, 50 mM Tris pH 9.6, NaCl 10.8gms, 5mM MgCl2)

4) Staining components: NBT (4-nitro blue tetrazolium chloride; 0.5 gms in 10mls 70% N,N-dimethylformamide) BCIP(5-bromo-4-chloro-3-indolyl-phosphate; 0.5 gms in 100% N,N-dimethylformamide)

Notes: BCIP (colorless) oxidation → forms blue precipitate. BCIP-NBT naturally forms this bluish purple precipitate over time; however, alkaline phosphatase speeds up the process 1000 fold. BCIP binds very tightly in the alkaline phosphatase active site, but when NBT reacts with BCIP, it is released from the enzyme and the colored precipitate forms.

Procedure: 1) Following blotting/spotting, immediately immerse the blot in excess blotto. Agitate for 5-10 minutes.

2) Pour off blotto and add limiting amounts – ‘cause it is expensive (!) (alkaline-phosphatase conjugated, anti-6X His) antibody at appropriate dilution. Agitate for 20 minutes.

3) Wash the blot 3 times, for 5 minutes each with blotto, and then with alkaline phosphatase buffer for 5 minutes.

4) Make up (5 mls) staining solution: to 5mls of alkaline phosphatase buffer, add 33 ul of NBT, and 17 ul of BCIP.

5) Pour off wash alkaline phosphatase buffer and add staining solution. Agitate and monitor for several minutes. When ready to stop the reaction, wash several times with water.

6) To record, photograph immediately, or store dry (though it will lose some color intensity).