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Staining total proteins on blot membrane with Ponceau S

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1. Background and purpose of the procedure

Total proteins transferred on blot membrane (either PVDF or nitrocellulose) can be determined by staining using Ponceau S dye. Staining is useful to ensure efficient transfer of proteins on membrane or to determine the position of non-pre-stained molecular weight ladder. Traditional Coomassie blue staining used on gel is inadequate for membrane as it will impair subsequent immunoblotting.

2. Materials and equipment

  • Blot membrane (nitrocellulose or PVDF)
  • Tris-buffered saline wash solution, TBS-T (see 3. Recipes)
  • Ponceau S staining solution (see 3. Recipes)
  • ddH2O
  • Flatbed scanner (if imaging of the stained membrane is required)
  • Bench rocker
  • Staining tray

3. Recipes

Tris-buffered saline (TBS) 10X

200 mM Tris-base (Fisher Bioreagents, Molecular Biology, BP152-500) [24.2 g for 1 L]
1.37 M Sodium chloride (Fisher Chemical, Certified ACS, S271-1) [80 g for 1 L]
ddH2O qsp 1 L 

Adjust pH to 7.6 using HCl (Fisher Chemical, Certified ACS Plus, A144-500)

Tris-buffered saline wash solution (TBS-T)

1/10 TBS 10X [100 mL for 1 L]
0.1% Tween 20 (Fisher Bioreagents, BP337-100) [1 mL for 1 L]
ddH2O qsp 1 L

This solution can be stored for up to 6 weeks at room temperature.

Ponceau S staining solution

0.1% Ponceau S dye (Fisher Bioreagents, BP103-10) [0.1g for 100 mL]
5% Acetic acid, glacial (Fisher Chemical, Certified ACS, A38-500) [5 mL for 100 mL]
ddH2O qsp 100 mL

This solution can be stored for several weeks at room temperature.

4. Methods

  1. Rinse the membrane twice with ddH2O to remove any residual SDS
  2. Add Ponceau S staining solution to the membrane and incubate under constant agitation on rocker for 10 min
  3. Remove and discard Ponceau S staining solution
  4. Rinse the membrane with three ddH2O washes of 2 min
  5. Capture image on flatbed scanner if desired
  6. Mark molecular weight ladder positions with pencil if needed
  7. Rinse the membrane with five TBS-T washes of 5 min on rocker
  8. Repeat washes with TBS-T until red staining completely disappear from membrane
  9. Membrane can be used for Western blotting (immunoblotting) using standard protocol

5. References

Litovchick L. Staining the Blot for Total Protein with Ponceau S. Cold Spring Harb Protoc. 2020 Mar 2;2020(3):098459. doi: 10.1101/pdb.prot098459. PMID: 32123012.

Protocol: Ponceau stain from Peter Pioli Lab at Western Michigan University School of Medicine

6 Revision history

Revision #DatePrepared by
1.02021-09-23Elie Besserer-Offroy
Summary of modifications
Initial version of the protocol

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