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  • 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.