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Western Transfer Protocol for Anti-MBP Monoclonal Antibody, HRP Conjugated |  | | Western Transfer Protocol for Anti-MBP Monoclonal Antibody, HRP Conjugated |
Overview
Materials Needed:
Transfer apparatus and associated buffers
Nitrocellulose or PVDF membrane
TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20)
Blocking Buffer (TBST + 5% Nonfat Dry Milk)
Anti-MBP Monoclonal Antibody, HRP conjugated, (NEB# E8038S)
Detection reagent
Introduction
The following protocol is for a 10 cm x 10 cm gel:
Protocol
- Transfer protein from the gel to a nitrocellulose or PVDF membrane following the directions of the transfer apparatus manufacturer. Mark the wells of the gel on the filter with a blunt pencil before removing and discarding the gel.
- Rinse the membrane with TBST.
- Incubate the membrane with Blocking Buffer for 1 hour at room temperature (or overnight at 4°C) with gentle shaking.
- Wash the membrane in 25 ml TBST at room temperature with gentle shaking, 3 times for 5 minutes each.
- Add 5 ul of the anti-MBP monoclonal, HRP conjugated, to 10 ml Blocking Buffer (a 1:2,000 dilution). Cover the membrane with the antibody dilution and incubate for 1 hour at room temperature with gentle shaking.
- Wash the membrane in 25 ml TBST at room temperature with gentle shaking, 3 times for 5 minutes each.
- Follow the manufacturer's directions for detection.
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