BCA Assay
What is the purpose of using a standard curve in determining protein concentration?
In this case, the greater the absorbance, the higher the protein concentration. Data for known concentrations of protein are used to make the standard curve, plotting concentration on the X axis, and the assay measurement on the Y axis. The same assay is then performed with samples of unknown concentration.
How do you find the unknown protein concentration of a standard curve?
Perform the assay and calculate the standard (see below). The result should be around 0.5mg/ml. To calculate the concentration of the undiluted, unknown sample, simply multiply by the dilution factor. So, 0.5 x 10= 5mg/ml
How do you use a standard curve to determine protein concentration?
Perform the assay and calculate the standard (see below). The result should be around 0.5mg/ml. To calculate the concentration of the undiluted, unknown sample, simply multiply by the dilution factor. So, 0.5 x 10= 5mg/ml
Why do we purify proteins?
Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.
How do you detect protein?
Specific methods which can detect amount of a single protein Spectrometry methods: High-performance liquid chromatography (HPLC): Chromatography method to detect proteins or peptides. ... Antibody dependent methods: Enzyme-linked immunosorbent assay (ELISA): Specifically can detect protein down to pg/mL.
BCA
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.
How does the BCA assay work?
The Bicinchoninic Acid (BCA) Protein Assay primarily relies on two reactions. Firstly, the peptide bonds in the protein sample reduce Cu2+ ions, in a temperature dependent reaction, from the copper solution to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution.
What is a BSA standard curve?
To know determine the actual concentration of a protein a standard curve is required. A standard curve is a plot of absorbance vs. a varying amount of some known concentration of protein. ... Two common proteins used for standard curves are bovine serum albumin (BSA) and an immunoglobin (IgG).
BCA assay
is used to determine the total level of protein in a solution.
