How do you prepare a standard curve?
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 does Bradford assay calculate protein concentration?
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.
How do you make a powder solution?
Mix with powdered compound until dissolved. For example: Mix 500 mL of water and 25 g of NaCl to make a 5% solution. Remember, if you’re diluting a liquid compound, you must subtract out the volume of liquid being added from the final volume: 500 mL – 25 mL = 475 mL of water.
What is the principle of Bradford assay?
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.
Which one of these is a limitation or disadvantage of the Bradford protein assay?
The biggest disadvantage of the Bradford protein assay is that it does not work if detergents or surfactants are in the sample or if the sample is basic. Particularly surfactants that are often used to solubilize some types of proteins will interfere with the test, causing the dye to precipitate out.
What is a 5% solution?
5% v / v solution means 5 ml of solute is dissolved 100 ml of solution.
Why is Bradford assay useful?
The Bradford assay for protein is widely used because of its sensitivity, speed, convenience, lack of need for a UV-capable spectrophotometer, and adaptability to 96-well plates. The “Bradford Reagent” is an acidic stain which turns blue when it interacts with protein.
How is the chromophore formed in Bradford assay?
A soluble dye chromophore is only formed above the working range of the assay indicating that precipitation of the dye by protein contributes to the assay mechanism.