What assay uses Coomassie brilliant blue?
the Bradford assay
The Coomassie brilliant blue stain is used in the Bradford assay, a colorimetric protein assay, to quantify the proteins separated by gel. This assay is performed by determining the absorbance shift of the Coomassie Brilliant Blue G-250.
What is the standard curve in a Bradford assay?
BSA and IgG are typical standards used to construct the curve. For BSA, use 0 – 1 mg/ml as your standard curve concentration; for IgG, use 0 – 1.6 mg/mL. Prepare a standard curve of absorbance versus micrograms protein and determine amounts from the curve.
Why is the wavelength of 595 nm used in measuring the absorbance of the standards and samples?
Higher absorbance of light at 595 nm means greater concentration of protein present in the sample. A spectrophotometer is an instrument that is designed to measure the amount of light which passes through a solution (spectro = range; photo = light; meter = tool for measuring).
How do you make BSA standards for Bradford assay?
Bradford Assay Materials: BSA standard solution (0.1 µg/µl) • Bradford solution o Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. Add 100 ml of 85% phosphoric acid while stirring continuously. When the dye has dissolved, dilute to 1 l in H2O.
Why is BSA used as standard for protein estimation?
BSA is used because of its ability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
How does a BCA assay work?
The BCA assay method is based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion generated by the biuret reaction under alkaline conditions. The bicinchoninic acid cuprous complex forms a deep blue color that is read at 562 nm, and the detection range is 0.2–50 μg.
Why BSA is used as a standard in protein assays?
How do you make a BSA standard?
Preparation of standard solutions
Bovine serum albumin (BSA) is frequently used as a reference protein. Prepare a BSA standard solution by dissolving precisely 100 mg of BSA (MB046) in a final volume of 10 mL of redistilled water.
Why is 595 nm used during a Bradford assay?
The difference between the two dye forms is greatest at 595 nm, making it the optimal wavelength to measure the blue color from the Coomassie dye–protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm.
Why is BSA used as standard?
How do you prepare BSA standards?
Why is BSA used as a standard?
How many kDa is BSA?
66 kDa
BSA is a 585 amino acids, 66 kDa globular protein comprised of at least 67% α-helical secondary structure in physiological conditions [15].
What is BCA protein assay protocol?
The BCA protocol requires a working solution mixed with the sample; when protein is present, the reaction produces a purple color that absorbs light at 562 nm and is quantified with a spectrophotometer. The BCA assay is similar to other protein quantification assays such as Lowry or Bradford assays.
What is the purpose of BCA protein assay?
BCA (Bicinchoninic Acid) Protein Assay. The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid.
Why is BSA a good standard?
Why is BSA used as a standard in Bradford?
The Bradford assay responses of the unmodified and reductively methylated proteins are summarized in Fig. 3. Typically, BSA is used as a standard for the Bradford assay, and a calibration curve based on the mass concentration of BSA is used to determine the unknown concentration of a protein.
How do you do a BSA assay?
Bradford Assay – YouTube
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. This requires that we consume only a tiny portion of each fraction. Set up a standard curve of protein dilutions in your plate.
What is standard protein solution?
A standard assay procedure, in which the concentrated dye reagent must first be diluted and filtered, is described for protein concentrations of 200-1400 µg/ml. A micro assay procedure is also included for much lower protein concentrations (1-20 µg/ml).
What is the size of BSA protein?
7.1 nm
Being the most abundant serum protein in bovine blood, which is a byproduct of the cattle industry, BSA is a relatively cheap and easily accessible compound. According to the literature, BSA has a nominal size of 7.1 nm and a molecular mass of 66.5 kDa.
How is BCA assay done?
The BCA assay requires two chemical reactions in order to produce the purple colored product that can be measured by spectrophotometry at 562 nm. The most common instrument used to measure this product is a plate reader, because a standard protein concentration curve is needed to produce a truly quantitative result.
What is BSA in a BCA assay?
Pierce BCA Protein Assay Protocol.. Materials. This is the protocol to be used in the determination of total protein in brain tissue by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and samples are prepared by sonification in 1% (w/v) SDS. REAGENT PREPARATION.
What is the difference between BCA and Bradford assay?
The key difference between BCA and Bradford assay is that BCA assay is time-consuming and less accurate, whereas Bradford assay is quick and accurate. BCA and Bradford assay are two assay methods of protein concentration determination.
Is BCA assay accurate?
It uses about the same amount of protein as the Lowry assay, gives fairly accurate results, and is the fastest and easiest to perform among the protein assays. It is also compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents.