Does Ponceau work on PVDF?
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.
How do you stain a PVDF membrane?
How to stain PVDF membrane?
- Soak blott after D.I. water rinse with methanol.
- Stain PVDF membrane with 0.2% Amido Black in 40% MeOH for 30 seconds to one minute.
- Destain in D.I. water with multiple changes until bands are clear and low background.
- Cut out band of interest.
What is Ponceau S and why do we use it during Western blotting?
Ponceau S is a rapid and reversible stain for detecting protein bands on Western blot membranes and can be used with PVDF, nitrocellulose and cellulose acetate membranes*. Since, Ponceau-S staining is reversible, it allows further immunological detection.
Can Ponceau stain be reused?
Do not reuse the stain; it will result in nonreproducible results because of depletion of the dye after the first use.
What is brilliant blue Ponceau?
Both Ponceau S and Coomassie Brilliant Blue stains are negatively charged solutions that bind to positively charged amino acid groups and non-polar protein regions. Ponceau S detects protein levels at 200 ng and higher, is compatible with PVDF, nitrocellulose, or nylon membranes.
Is Coomassie blue or Ponceau more sensitive?
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.
What is the difference between PVDF and nitrocellulose membranes?
PVDF membranes have a higher protein binding capacity than nitrocellulose. The protein binding capacity of PVDF ranges from 150-200 µg of protein/cm2 and nitrocellulose ranges from 80-100 µg of protein/cm2. Although PVDF has a higher binding capacity, it could result in increased background in some circumstances.
How do you store PVDF membrane after blotting?
Place the sandwich between two sheets of card stock or thin cardboard. Use paperclips to clip the stack together on the edges. Place the stack in a plastic bag and seal the plastic bag closed. Store the blot at 4˚C for up to 2 weeks, -20˚C for up to 2 months, or -70˚C for longer storage.
What is Ponceau S staining?
Ponceau S is a negatively charged, red colored stain which binds to positively charged amino groups and non-polar regions of proteins. It has a detection limit of around 250 nanograms of protein following SDS-PAGE and electrotransfer to nitrocellulose membranes (1).
Is Ponceau S stain is a method of total protein normalization or housekeeping protein normalization?
Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization.
How to use Ponceau S staining solution for protein electrotransfer?
1. Following protein electrotransfer, rinse membrane briefly in ddH 2 O to remove any detergent that may inhibit staining. 2. Incubate membrane in Ponceau S Staining Solution for 5-10 minutes at room temperature. 3. Wash membrane in ddH 2 O until distinct reddish-pink protein bands are visible (1-5 min).
How do you stain A Ponceau S membrane?
Incubate membrane in Ponceau S Staining Solution for 5-10 minutes at room temperature. 3. Wash membrane in ddH 2 O until distinct reddish-pink protein bands are visible (1-5 min).
What is the best method of staining PVDF?
All conventional methods of staining may be employed with PVDF membranes with slight modifications as below. Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE usually 15 to 20 seconds will suffice.
How do you stain a membrane after protein electrotransfer?
1 Following protein electrotransfer, rinse membrane briefly in ddH 2 O to remove any detergent that may inhibit staining. 2 Incubate membrane in Ponceau S Staining Solution for 5-10 minutes at room temperature. 3 Wash membrane in ddH 2 O until distinct reddish-pink protein bands are visible (1-5 min).