Home Protocols Western Transfer Protocol for Anti-MBP Monoclonal Antibody

Western Transfer Protocol for Anti-MBP Monoclonal Antibody

Overview

Materials Needed:
Transfer apparatus and associated buffers
Nitrocellulose or PVDF membrane
TBST (20 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20)
Blocking Buffer (TBST + 5% Nonfat Dry Milk)
Anti-MBP Monoclonal Antibody (NEB #E8032S )
Anti-Mouse Antibody conjugated to peroxidase
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 25 ml Blocking Buffer for 1 hour at room temperature (or overnight at 4°C) with gentle shaking.
  4. Wash the membrane in 25 ml TBST for 5 minutes with gentle shaking, 3 times for 5 minutes each.
  5. Add 1 ul of the anti-MBP monoclonal antibody to 10 ml Blocking Buffer (a 1:10,000 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. Make a dilution of the Anti-Mouse IgG-peroxidase conjugate in 10 ml Blocking Buffer according to the manufacturer's recommendation, and incubate the membrane in the solution for 1 hour.
  8. Wash the membrane in 25 ml TBST at room temperature with gentle shaking, 3 times for 5 minutes each.
  9. Follow the manufacturer's directions for detection.