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

  1. 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.
  2. Rinse the membrane with TBST.
  3. Incubate the membrane with Blocking Buffer for 1 hour at room temperature (or overnight at 4°C) with gentle shaking.
  4. Wash the membrane in 25 ml TBST at room temperature with gentle shaking, 3 times for 5 minutes each.
  5. 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.
  6. Wash the membrane in 25 ml TBST at room temperature with gentle shaking, 3 times for 5 minutes each.
  7. Follow the manufacturer's directions for detection.