How does simply blue stain work?
SimplyBlue Safe Stain is a ready-to-use, fast, sensitive, and safe Coomassie G-250 stain for visualizing protein bands on polyacrylamide gels and on dry PVDF membranes. This is completely non-hazardous and does not require methanol or acetic acid fixatives or destains.
What is Coomassie blue 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 make Coomassie stain?
Stain: Dissolve 0.4g of Coomassie blue R350 in 200 mL of 40% (v/v) methanol in water with stirring as needed. Filter the solution to remove any insoluble material. Add 200mL of 20% (v/v) acetic acid in water. The final concentration is 0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol, and 10% (v/v) acetic acid.
Can you Coomassie stain overnight?
After incubation, discard the stain. Stain cannot be re-used. Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity will decrease. If you need to leave the gel overnight in the stain, add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain.
When should I use silver stain?
Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals.
Does Coomassie Blue stain all proteins?
The most common used protein stain is Coomassie Blue staining, which is based on the binding of Coomassie Brilliant Blue, which binds non-specifically to virtually all proteins.
Why does Coomassie Blue bind to protein?
When proteins bind to Coomassie blue in acid solution their positive charges suppress the protonation and a blue colour results. The binding of the dye to a protein causes a shift in the absorption maximum of the dye from 465 to 595 nm and it is the increase in absorbance at 595 nm that is monitored.
What is the difference between Coomassie R250 and G-250?
The R-250 (red-tinted) form lacks two methyl groups that are present in the G-250 (green-tinted) form, which is also called colloidal coomassie dye. Typically, coomassie gel stains and protein assay reagents are formulated as very acidic solutions in 25 to 50% methanol.
What is the purpose of Coomassie staining?
Can you transfer a gel after Coomassie?
Normal staining with coomassie requires fixing the gel, so you will not get transfer. You can stain your gel after transfer.
What is the advantage of silver staining?
The protein detection by silver staining is a highly sensitive method yet specific and selective for proteins. It produces an image with reduced background and less mass spectrometry interference.
What is the purpose of a silver stain?
Silver staining is an excellent technique for detecting proteins which are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its efficiency of detecting proteins present in nanograms.
Why do we use Coomassie Blue?
What does Coomassie Blue bind to?
In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine).
What does a Coomassie stain tell you?
The Coomassie stain can interact with a small group of amino acids: arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan making it a useful stain for fingerprint analysis to identify the biological sex of the fingerprint originator.
Can one use Commassie brilliant blue R 250 in Bradford’s reagent?
The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins.
How does Coomassie G 250 interact with proteins?
In acidic conditions, the dye binds to proteins primarily through basic amino acids (primarily arginine, lysine and histidine), and the number of coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.
How does Coomassie bind to proteins?
Can I stain Coomassie overnight?
Coomassie blue staining of proteins
Submerge the gel in the Commassie blue stain solution. Use just enough to completely submerge the polyacrylamide gel. Stain for 1-4 hours or overnight at room temperature with gentle shake. Coomassie blue stain solution can be reused for serveral times.
How long should I stain gel?
Incubate at room temp with gentle shaking for 10-15 min. Heating allows the gel to stain faster. Alternatively, soak gel in stain for 1 hr at room temperature.
What is the principle of silver staining?
The technique is based on the simple principle that selective reduction of silver into metallic silver occurs at the initiation site in the close proximity of protein molecules. The staining process sequentially consists of protein fixation, sensitization, washing, silver impregnation, and finally development of image.
Which type of detection is used for silver stain?
Silver staining is the most sensitive colorimetric method for detecting total protein. The technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups.
Is silver staining toxic?
Yes, silver stain is highly toxic. Care must be taken while using and disposing the stain. Silver stain should never be thrown into the sink. It must be disposed of properly in an appropriate waste container using the recommended procedure.
How does Coomassie Blue bind protein?
Why is Coomassie Blue a good stain for proteins?
The most common method for in-gel protein detection is staining with Coomassie blue dye. Coomassie dye staining is especially convenient because it involves a single, ready-to-use reagent and does not permanently chemically modify the target proteins.