Can you Coomassie stain a PVDF membrane?
Coomassie R-250: 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. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage)
Can you do a western after Coomassie?
The answer is yes: western blotting Coomassie-stained proteins can be done, but it’s not a simple or efficient process. As you know, there are two types of Coomassie stains – “classical” and “colloidal”. Proteins stained by one of these two methods will behave differently if you try to blot them afterwards.
Can you stain membrane with Coomassie?
BASIC PROTOCOL 2: COOMASSIE BLUE R-250 STAINING Coomassie blue R-250 can be used with most types of blot membranes except nitrocellulose (high concentrations of organic solvents can dissolve nitrocellulose membranes).
How do you stain a Western blot membrane?
Staining method
- Place the blot transfer membrane in a plastic box and rinse it with water three times, 5 minutes each.
- Stain the membrane with Ponceau S stain for 30 seconds to 1 minute.
- Destain the membrane with several changes of water for 30 seconds to 1 minute each, then air dry.
What is Coomassie stain used for?
Description. Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (“destaining”). This treatment allows the visualization of proteins as blue bands on a clear background.
What is colloidal Coomassie?
Bio-Rad’s QC colloidal Coomassie stain is a ready-to-use single-bottle protein stain that does not require the mixing of any components or addition of any alcohols. It is a special formulation of Coomassie G-250 that provides maximum sensitivity with low background for a wide variety of acrylamide gel chemistries.
What is Coomassie staining?
Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (“destaining”). This treatment allows the visualization of proteins as blue bands on a clear background.
How do you remove Coomassie gel stain?
The Coomassie stain is removed by aspiration after staining. 4. Cover the gel with ~250mL of the destain solution and allow the gel to destain with gentle agitation. The destain solution should be changed several times, removing it at each change by aspiration.
How does Coomassie stain bind to proteins?
In the staining reaction, the Coomassie dye binds to proteins through ionic interactions between sulfonic acid groups and positive protein amine groups through Van der Waals attractions.
What is Western blot used for?
Western Blot Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis to separate the sample’s proteins. The separated proteins are transferred out of the gel to the surface of a membrane.
Is Coomassie staining necessary after transfer in western blotting?
In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer.
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 much protein can be detected in a Coomassie stain?
3-10 microgram of protein can be detected usually within 5 minutes in Coomassie stains. With additional water-based de-staining, as little as 7 ng of protein (BSA) can be detected.
What is the best solvent for Coomassie staining?
The classical Coomassie treatment involves incubating the gel in a mix of 40% methanol and 10% acetic acid (the solvent for the Coomassie stain), which should, theoretically, interfere with transfer.
https://www.youtube.com/watch?v=7SVHqK_mFtQ