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Protein & proteomics

Stripping and reprobing a western blot

Remove bound antibodies with a mild glycine or a harsh SDS/β-mercaptoethanol buffer and probe the membrane again.

Total time1–1.5 h
Hands-on30 min
LevelBasic
Steps5
0 of 5 steps done

Before you start

  • Strip PVDF rather than nitrocellulose when possible (more robust).
  • Probe the weakest or most important target first.

Reagents and materials

Totals are for 1 membranes (+10% extra). Per-membrane amounts are in the middle column.

ReagentPer membraneTotal
Stripping buffer20 mL20 mL
PBS / TBST (washes)80 mL80 mL
Blocking buffer15 mL15 mL

Buffers and recipes

Mild stripping buffer per 1 L

Glycine15 g
SDS1 g
Tween-2010 mL
Ultrapure waterto 1 L

Adjust to pH 2.2.

Harsh stripping buffer per 100 mL

1 M Tris-HCl, pH 6.8 (62.5 mM)6.25 mL
10% SDS (2%)20 mL
β-Mercaptoethanol (~100 mM)0.7 mL
Ultrapure waterto 100 mL

Add β-mercaptoethanol in a fume hood just before use.

Equipment

  • Rocker
  • Fume hood and 50 °C water bath (harsh method)

Procedure

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

  1. Mild strip

    Incubate in mild stripping buffer 2 × 10 min at room temperature.

  2. Or harsh strip

    Incubate in harsh buffer at 50 °C for 30–45 min with agitation in a fume hood.

  3. Wash

    Wash 2 × 10 min PBS, then 2 × 5 min TBST (longer for the harsh method).

  4. Confirm stripping

    Incubate with ECL and image to confirm the old signal is gone (or re-probe with secondary only).

  5. Re-block and re-probe

    Block for 1 h and continue with the new primary antibody.

Critical points

  • Stripping removes some antigen — do not compare band intensities before and after stripping.

Troubleshooting

ProblemWhat to check or change
Old signal remainsUse the harsh method or longer incubation.
Weak new signalAntigen loss: run a parallel blot instead.

Safety

β-Mercaptoethanol is toxic and malodorous: fume hood only.

Further reading

  • Abcam western blot stripping protocol (technical resource).

This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.