What is the purpose of the standard curve in a Bradford assay?
The traditional method for calculating protein concentration of an unknown sample is to use a standard curve that is generated from known protein standards. The most reliable protein estimation is performed using a reference or a protein standard that has properties similar to the protein being estimated.
What happens when Coomassie Brilliant Blue binds to protein?
When proteins bind to Coomassie blue in acid solution their positive charges suppress the protonation and a blue colour results. The binding of the dye to a protein causes a shift in the absorption maximum of the dye from 465 to 595 nm and it is the increase in absorbance at 595 nm that is monitored.
What is a standard curve in protein assay?
Standard curves represent the relationship between two quantities. They are used to determine the value of an unknown quantity (glucose concentration) from one that is more easily measured (NADH level). An example of a standard curve for protein concentration determination is illustrated in Figure 5-1.
What assay uses Coomassie brilliant blue?
The Bradford Assay
The Bradford Assay: Colorimetric Protein Determination with Coomassie Blue. The Bradford assay is based on the use of the dye Coomassie Brilliant Blue G-250, which is frequently abbreviated as Coomassie G-250 or Coomassie Blue. This is one of two Coomassie dyes that are often confused.
How does BCA assay calculate protein concentration?
You can use this formula to calculate the amount of protein in your sample: [ (OD of the test sample) / (OD of the standard sample) ] X concentration of the standard sample. = 4.0 mg/mL protein (in test sample).
When plotting a standard curve for a protein assay What is your independent variable?
The standard curve will illustrate the relationship between concentration (the dependent variable) and absorbance at 540 nm (the independent variable).
What does a standard curve tell you?
A standard curve is a tool that allows us to estimate the DNA concentration of unknown samples by comparing them to standards with known DNA concentrations.
What is the standard curve equation?
The equation y=mx+b can be translated here as “absorbance equals slope times concentration plus the y-intercept absorbance value.” The slope and the y-intercept are provided to you when the computer fits a line to your standard curve data. The absorbance (or y) is what you measure from your unknown.