Western Transfer Protocol for Anti-MBP Antiserum
Overview
Materials Needed:
Transfer apparatus and associated buffers
Nitrocellulose or PVDF membrane
TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20)
Blocking Buffer (TBST + 5% Nonfat Dry Milk)
Anti-MBP antiserum (NEB# E8030S)
Anti-rabbit antibody conjugated to peroxidase
Detection reagent
Introduction
Follow the following steps for a 10 cm x 10 cm gel:
Protocol
- Transfer the 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 25 ml Blocking Buffer for one 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 1 μl of the anti-MBP antiserum to 10 ml Blocking Buffer (a 1:10,000 dilution). Cover the membrane with the antibody dilution and incubate for one 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.
- Make a dilution of an anti-rabbit IgG-peroxidase conjugate in 10 ml Blocking Buffer according to the manufacturer's recommendation, and incubate the membrane in the solution for one hour.
- 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.
