Ponceau S Stain Protocol

Introduction

Ponceau S staining is a rapid and reversible staining method used for the detection of protein bands on Western blot membranes, Polyvinylidene fluoride (PVDF), nitrocellulose, and cellulose acetate membranes. Ponceau S is a negative stain that binds to the positively charged functional groups of the protein (amino group) and the non‐polar regions of the protein. The method could also be used to measure the microgram quantities of transferred protein by obtaining the reddish pink protein bands with a clear background. The Ponceau S stain is reversible; this quality makes it useful for further immunological detection.

Ponceau S stain is useful to locate and identify the proteins transferred electrophoretically onto nitrocellulose/nylon membranes before the antibody-mediated detection as the dye does not interfere with antibody detection in Western blots. It has also been used as an alternative to a loading control in determining the levels of protein expression for semiquantitative immunoblotting. Proteins stained with Ponceau S and solubilized in dimethylsulfoxide could be detected at 529 nm.

Properties

• Chemical formula: C₂₂H₁₆N₄O₁₃S₄
• Molar mass: 672.63 g·mol⁻¹

Protocol (Goldring., 2015)

Materials

• Blot transfer membrane
• Ponceau S stain: 0.5% (w/v) Ponceau S dissolved in 1% (v/v) acetic acid
• 200 μM NaOH/20% (v/v) acetonitrile
• Plastic box

Staining method

1. Place the blot transfer membrane in a plastic box and rinse it with water three times, 5 minutes each.
2. Stain the membrane with Ponceau S stain for 30 seconds to 1 minute.
3. Destain the membrane with several changes of water for 30 seconds to 1 minute each, then air dry.

Do not over-destain, as the protein bands would be challenging to detect.

Note: Stop destaining as soon as the background gets a slight pink tinge.

4. Photograph the blot to have a permanent record of the staining pattern.
5. Remove the Ponceau S stain from the protein bands using 200 μM NaOH/20% acetonitrile for 1 minute.
6. Wash the membrane three times with water for 5 minutes each time, then air dry it.

Procedure for cellulose acetate total protein detection

1. Immerse the cellulose acetate membrane in Ponceau S staining solution for 5 minutes.
2. Then, immerse the membrane in 10% acetic acid (v/v) aqueous solution for 5 minutes, change the aqueous solution, and again immerse the membrane for 5 minutes.
3. Transfer the membrane to methanol and soak it for 5 minutes.
4. Transfer the membrane to a clearing solution (methanol: acetic acid: polyethylene glycol in a ratio of 70:30:4 by volume) for 5 minutes.
5. Remove the membrane from the clearing solution and dry it.

Applications

An alternative to housekeeping genes as a loading control (Romero-Calvo. et al., 2010)

For the quantification of proteins in a tissue, a ‘housekeeping protein’ with relatively constant expression in the tissue is used as an internal loading control. β-actin and glyceraldehyde 3-phosphate dehydrogenase (GADPH) have been initially used as loading controls, but because of their participation in other cellular processes, Ponceau S stain was used as an alternative. Lower cost, fast staining time, and reversible nature made the Ponceau S stain superior over the housekeeping genes. The results showed that the Ponceau S stain serves as a better loading control as compared to the β-actin.

Measurement of total protein in the urine (Meola., Vargas., & Brown., 1977)

Ponceau S stain was used to measure total protein in the urine. The method is simple, sensitive, and free of any interference from drugs as in other methods. The coefficient of variation (CV) for a 1.1 g/liter urine control was 4.6%. Firstly, the proteins were adsorbed onto cellulose powder, then the Ponceau S dye was added to the protein. The dye was eluted in NaOH after washing away the excess dye.  The staining technique showed better results as compared to the biuret and turbidimetric methods. Also, it enabled the measurement of a broader range of proteins than the antibody-based immunological methods.

Protein determination on nitrocellulose membranes (Bannur. et al. 1999)

In the study, the Ponceau S stain was used to estimate protein concentrations using color image analysis of protein spots. The method involved the identification of a constant volume (2 ml) of the protein solutions on nitrocellulose paper, stained with acidic ponceau S. The image of the nitrocellulose paper was taken with the help of a digital color scanner. The intensity of the color in the spot was measured as inverse integrated gray value. A discernible increase in protein concentration from 0.1 to 50 mg protein per spot was observed. The method was found simple, easy, and allowed simultaneous analysis of several samples.

Precautions

• Avoid prolonged or repeated exposures
• The Ponceau S stain can be used on most membranes including PVDF (polyvinylidene difluoride) except nylon membranes.
• Destaining should be performed using 10% acetic acid.
• If nitrocellulose membranes are used, then destaining should not be done with alcohol.
• Rinse the membrane with 0.1 M NaOH to remove the dye from the protein for further immunochemical testing.

Strengths and limitations

• Ponceau S stain enables the detection of proteins even at lower concentrations such as ranging from 1–10 μg protein.
• The staining method is rapid, sensitive, cheap, reversible, and easy-to-use.
• Ponceau S, followed by complete destaining, does not interfere with the subsequent analytical procedures enabling further immunoblotting assays.
• The method allows the isolation of pure protein; therefore, avoiding elaborate procedures required for protein recovery from polyacrylamide gels.
• On the neutral membranes, the stain can be easily removed from proteins by washing with distilled water or Tris-buffered saline.
• Easy removal following excessive washing and less sensitivity limits the use of Ponceau S stain in protein identification from gels.

References

1. , P. J. (2015). Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose. Methods Mol Biol, 1314, 19-25.
2. I. Romero-Calvo., B. Ocón., P. Martínez-Moya., D. M. Suárez., A. Zarzuelo., O. Martínez-Augustin., & Medina., S. F. (2010). Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem, 401(2), 318-320.
3. J. Meola., A. M. Vargas., & Brown., H. H. (1977). Simple procedure for measuring total protein in urine. Clin Chem, 23(6), 975-977.
4. S. Bannur., V. S. Kulgod., S. S. Metkar., K. S. Mahajan., & Sainis., K. J. (1999). Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes. Anal Biochem, 267(2), 382-9.