Can Ponceau staining be used as loading control?
In conclusion, we have shown that reversible Ponceau S staining can be used advantageously over actin detection for quality or equal loading control in Western blotting. It is equally useful for this purpose, has similar or improved dynamic range, is extremely inexpensive, and takes no longer than 10 min.
What is the purpose of Ponceau staining?
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.
Which is more sensitive Coomassie blue or Ponceau S?
Coomassie brilliant blue R250 and amido black 10B are more sensitive than Ponceau S2. However with Coomassie brilliant blue R250 the background staining is high and also this stain is not removed easily. Coomassie Brilliant Blue also interferes with immonodetection process.
How do you use the loading control in Western blot?
Use internal loading controls that are stably expressed and are minimally affected by experimental conditions. Select a loading control antibody against a protein known to be constitutively expressed in your sample. Utilize a second loading control to substantiate results obtained for the first control.
How do you remove Ponceau stains?
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.
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.
How do you prepare Ponceau stain?
Preparation of ponceau S staining solution
- For the preparing 500 ml of ponceau S staining solution add 25 ml of glacial acetic acid to 400 ml of distilled water.
- Add 0.5 gm of ponceau S tetrasodium salt to the acetic acid prepared above.
- Stir the mixture to dissolve.
- Make up the solution to 500 ml using distilled water.
Why would someone use Ponceau S instead of AB HRP color development or Coomassie?
Ponceau S staining protocol takes about 20 minutes, is non-toxic, and a gentler solution than Coomassie Brilliant Blue. Ponceau S staining solution does not fix the protein, allowing for western blot analysis after staining, which is another key consideration.
What is a loading control antibody?
Loading Control: A loading control is an antibody specific for a ubiquitously and constitutively expressed protein and can be used to normalize protein levels in Western blot. Loading control antibodies help in assessing that samples have been loaded equally across a gel.
How does a loading control work?
Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren’t due to loading or protein transfer errors.