What is the purpose of staining with Ponceau Red?

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.

What should you be able to see on your gel after staining with Ponceau Red?

What should you be able to see on your gel after staining with Ponceau Red? When performing the Ponceau Stain, you may see a band of about 25 kD in your negative control that is not present in your positive control or your other positive clones.

Can you stain with Ponceau after blocking?

staining the membrane with Ponceau only makes sense before blocking has been done; Ponceau is a non-specific protein stain and will visualize all proteins including the ones used for blocking (e.g. BSA or casein).

Which is more sensitive Coomassie blue or Ponceau S?

Ponceau S detects protein levels at 200 ng and higher, is compatible with PVDF, nitrocellulose, or nylon membranes. The traditional Coomassie Brilliant Blue is only compatible with PVDF membranes, but can detect protein levels at 50 ng and higher.

How does Ponceau stain work?

Ponceau S is a negative stain, which binds to the positively charged amino groups of the protein and it also binds non-covalently to non-polar regions in the protein. Microgram quantities of transferred protein can be detected with Ponceau-S Stain, which generates reddish pink protein bands with a clear background.

Does Ponceau interfere with antibody binding?

Although the Ponceau stain should not block antibody binding, you can take a picture of the Ponceau stained membrane, and then remove the Ponceau by soaking in a large volume of water for a few minutes.

Is Ponceau light sensitive?

*Note: Ponceau S is light sensitive.

How many times can you reuse Ponceau stain?

Rinse the membrane in distilled water until the background is clean. The stain can be completely removed from the protein bands by continued washing. Stain solution can be re-used up to 10 times.

Can you reuse Ponceau?

Do not reuse the stain; it will result in nonreproducible results because of depletion of the dye after the first use.

How do you remove Ponceau stain?

Completely submerge with Ponceau stain and put on rocker ▪ Nitrocellulose membranes saturate in ~5 min. PVDF membranes saturate in ~15 minutes. 3. Remove Ponceau from non-protein parts of the membrane via gentle rinsing with distilled H2O.

How do I remove Ponceau?

6. Completely remove Ponceau with 4-5x room temperature washes (~5-10 minutes per wash) ▪ Using 1x TBS/0.2% Tween-20. 7. Once all the “red” is gone, membrane can be blocked and probed as per normal procedure.

What is the best concentration of Ponceau S for staining?

According to Sander et al., 2019, the staining method works well regardless of the concentration of Ponceau S (2%-0.001%), acetic acid (1%-20%), or other acid types [4]. Therefore, a more dilute solution can also be used 0.01 % of dye and acetic acid (1%). We are providing other types of recipes used at the end of the post.

Does Ponceau S concentration affect the sensitivity of protein detection?

Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 – 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant.

What is the standard concentration of Ponceau S in acetic acid?

Our research suggests a standard concentration of 0.01% (w/v) Ponceau S in 1% (v/v) acetic acid used for 2 mins staining could be adopted by the scientific community to increase the rigor and reproducibility of Western blotting normalization. This work was partly funded by AHA 16GRNT31350040 and NIH 5P42ES004699.

Is Ponceau S toxic to humans?

Though Ponceau S is considered non-toxic to humans in small concentrations, the effect of being exposed to larger amounts of Ponceau S over time is unknown. Using less of any compound, even non-toxic, is good for the environment.