How do you calculate the concentration of a protein assay?
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 find the protein concentration from absorbance Bradford assay?
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.
Which assays are used to determine protein concentration?
Some of the protein assays currently being used in laboratories include the Lowry assay, the Bradford assay, the BCA assay and UV absorbance at 280 nm. Of these methods, the UV absorbance is by far the simplest and the most direct.
How does the BCA assay measure protein concentration?
The BCA Protein Assay combines the protein-induced biuret reaction (see above) with the highly sensitive and selective colorimetric detection of the resulting cuprous cation (Cu1+) by bicinchoninic acid (BCA). The purple colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion.
How do you calculate protein concentration in mg ml?
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.) Pure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.
How do you find protein concentration from absorbance?
Use the following formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm. Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.) Pure protein of known absorbance coefficient.
Which method is best for protein estimation?
The simplest and most direct assay method for protein concentration determination in solution is to measure the absorbance at 280 nm (UV range). Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption.
What other approaches can be used to determine protein concentration?
Different methods of estimation of Protein concentration are mentioned below:
- Biuret method: Sensitivity of this method is very low.
- UV absorption: Sensitivity of this method is moderate.
- BCA assay: This method is highly sensitive and detects proteins at a low concentration of 1 µg.
What is a BCA assay used for?
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.
What is the principle of BCA?
The principle of the bicinchoninic acid (BCA) protein assay1 relies on the formation of a Cu2+-protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the amount of protein present.