Below is a table prepared by a biochem student to construct a standard curve for
ID: 823657 • Letter: B
Question
Below is a table prepared by a biochem student to construct a standard curve for protein analysis. The Bradford assay was used with Bovine Serum Albumin (BSA, 0.1 mg/mL) as the standard protein. Complete the table by filling in the mass of the BSA samples and the approximate A595 that will be obtained for each tube. Assume the procedure was conducted correctly.tube no.
Reagent 1 2 3 4 5 6
Water (mL) 1.0 0.9 0.8 0.6 0.2 -- BSA Volum (mL) -- 0.1 0.2 0.4 0.8 1.0 BSA Weight (?g) Bradford Reagent (mL) all tubes contain 5.0 5.0 5.0 5.0 5.0 5.0 Abs @ 595 nm 0.00 0.08
What does the term BCA mean and what is the chemical basis for the determination of protein concentration by the BCA method?
Below is a table prepared by a biochem student to construct a standard curve for protein analysis. The Bradford assay was used with Bovine Serum Albumin (BSA, 0.1 mg/mL) as the standard protein. Complete the table by filling in the mass of the BSA samples and the approximate A595 that will be obtained for each tube. Assume the procedure was conducted correctly.
tube no.
Reagent 1 2 3 4 5 6
Water (mL) 1.0 0.9 0.8 0.6 0.2 -- BSA Volum (mL) -- 0.1 0.2 0.4 0.8 1.0 BSA Weight (?g) Bradford Reagent (mL) all tubes contain 5.0 5.0 5.0 5.0 5.0 5.0 Abs @ 595 nm 0.00 0.08 Water (mL) 1.0 0.9 0.8 0.6 0.2 -- BSA Volum (mL) -- 0.1 0.2 0.4 0.8 1.0 BSA Weight (?g) Bradford Reagent (mL) all tubes contain 5.0 5.0 5.0 5.0 5.0 5.0 Abs @ 595 nm 0.00 0.08
What does the term BCA mean and what is the chemical basis for the determination of protein concentration by the BCA method?
What does the term BCA mean and what is the chemical basis for the determination of protein concentration by the BCA method?
Explanation / Answer
The term BCA refers to the Bicinchoninic Acid Assay which is almost exclusively used for the quantitative estimation of protein concentrations. It is also commonly known as the Smith assay. This protein assay has higher efficiency than other common protein assays such as Bradford and Lowry.
The protein content in this assay is estimated colorimetrically by a colour change from green to purple. This colour change is directly proportional to the protein concentration. The other components of this assay apart from Bicinchoninic acid are:
- Sodium carbonate
-Sodium bicarbonate
- Sodium tartarate
- Cupric sulfate pentahydrate
The principle of the colourimetric protein estimation in this assay is quite simple. Initiallly the peptide bonds of proteins reduce the Cu2+ ions from cupric sulfate to Cu+ ions. This reduction reaction is directly proportional to the amount of protein present. After this, two molecules of bicinchoninic acid chelate to each Cu+ ion giving a purple coloured adduct which can then be colourimetrically quantified. Thus, these two simple reactions enable protein estimation in the BCA assay.
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