Stripping Western Blot Membranes

Written by Super User. Posted in Protein

Find information on how to strip western blot membranes here.

Western Blot Membrane Stripping Table of Contents

What is Membrane Stripping?

Membrane stripping is the removal of antibodies (including primary and secondary) from a western blot membrane, to allow the incubation of new primary and secondary antibodies for the assay of a new protein by western blotting.

Applications of Western Blot Stripping

Agarose gel electrophoresis is mainly used in analysis or separation of DNA and RNA molecules (although proteins can also be separated on agarose gels), however separation also allows the purification of specific sizes of DNAs after restriction enzyme digestion. This is usually used in cloning to obtain cut plasmids, in which agarose gel electrophoresis separates cut vectors from uncut ones.

Thus stripping allows:

  1. The reuse of western blot membranes to assay for another protein's expression
  2. To assay for total protein
  3. To assay for the non-phosphorylated protein (after assaying for phosphorylated protein).

 

 

Membrane Stripping Buffers

Stripping requires special buffers and incubation in an oven.

Buffers Used:

Stripping Buffer

  • 62.5 mM Tris-HCl (pH 6.8)
  • 2% SDS. (add 280 ul of 2-beta merceptoethanol or a final concentration of 0.1 M 2-Mecaptoethanol).

Stripping Protocol

  • add 280 μl of 2-beta-mercaptoethanol per 40 mL of stripping buffer (to a final concentration of 100 mM)
    1. Wash membrane in TBST (1 X 5 min)
    2. Incubate membrane in stripping buffer for 30 min at 50-60°C (use a heating oven for this - and keep the oven door closed due to smell)
    3. Wash in TBST (2 X 5 min)
    4. Block membrane for 1 h in 5% blocking milk
    5. Follow normal protocol for developing western blots.
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