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  • 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

    1. 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.
    2. Rinse the membrane with TBST.
    3. Incubate the membrane with 25 ml Blocking Buffer for one 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 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.
    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 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.
    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.