What is the minimum and maximum protein concentration that the Bradford assay can detect?
The assay can detect 0.005 mg/ml, so we’ll dilute each fraction 100-fold, making 0.5 mg/ml in the undiluted fraction the minimum we can reliably detect.
How do you calculate protein concentration from absorbance 595?
Determine the best fit of the data to a straight line in the form of the equation “y = mx + b” where y = absorbance at 595 nm and x = protein concentration. Use this equation to calculate the concentration of the protein sample based on the measured absorbance.
What does Bradford assay measure?
The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue.
What is another name for Coomassie blue?
Coomassie Brilliant Blue G
| PubChem CID | 61363 |
|---|---|
| Structure | Find Similar Structures |
| Chemical Safety | Laboratory Chemical Safety Summary (LCSS) Datasheet |
| Molecular Formula | C47H48N3NaO7S2 |
| Synonyms | Brilliant Blue G 6104-58-1 UNII-M1ZRX790SI Brilliant blue G-250 M1ZRX790SI More… |
What is the purpose of SDS PAGE?
SDS-PAGE is an analytical technique to separate proteins based on their molecular weight. When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix.
Why do we use SDS PAGE instead of agarose gel electrophoresis for proteins?
DNA is a high molecular weight molecule.It need pore size should be high compare to PAGE. For protein in SDS gel, we normally run longer time right. If you use agarose gel, it will melt before your getting your results. Sometimes, at the end of SDS page running time, you may notice the running solution is hot.
What did we use to stain the proteins on our SDS PAGE gel?
Coomassie Blue stain is used to stain the protein bands in polyacrylamide gels. One common way to use it is to dissolve the dye in a mixture of methanol, acetic acid, and water. This stain will permeate the gel, stain the protein, and also fix the protein in place.
Why is Coomassie blue used in SDS-PAGE?
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.
Can you silver stain after Coomassie?
Silver staining over Coomassie-stain can work, but is usually quite ugly. If you can see your bands (even faintly) on Coomassie, I would run a separate gel with 10-30% sample and silver stain from fresh – it is about 10-100x more sensitive.
What is silver stain used for?
Silver staining is a special yet powerful staining technique that is used for the detection and identification of proteins in gels. This is because silver binds to the chemical terminal or side chains of amino groups i.e carboxyl and sulfhydryl groups.
How does a silver stain work?
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.
Why does silver nitrate stain skin?
Silver nitrate stains appear black or gray on the skin. Silver nitrate is a chemical substance that is used to develop photographs and in some medical procedures. If the substance comes into contact with the skin, it leaves a residue which will gradually darken to a black or gray color over several hours.
How sensitive is silver staining?
0.25-0.5