3.4: Quantitative Detection of Protein (Activity)
- Page ID
- 24748
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Experimental Background
Bovine Serum Albumin (BSA) is a protein that circulates in the blood of cows. Purified BSA can be used with Biuret solution in serial dilutions to generate a Standard Curve. The standard curve will illustrate the relationship between concentration (the dependent variable) and absorbance at 540 nm (the independent variable). We can then use this curve to estimate the concentration of unknown samples.
1. On a graph, do you remember which axis is the dependent and which is the independent variable?
2. In the table below, can you identify which samples are the negative controls and which are the positive controls?
3. What is the prediction of the absorbance or color intensity of the different tubes?
Dilute BSA Standards
- Label 9 tubes 1-9.
- Combine the components of the table below to generate the appropriate concentration of solutions.
- Place tube 1 (blank) into a cuvette and measure the absorbance (A) in the spectrophotometer at 540 nm.
- Calibrate the spectrophotometer to read 0 at A540nm.
- Sequentially read each sample at A540nm and record values in the table below.
- Plot each BSA dilution in a spreadsheet program like Excel as a scatterplot.
- Generate best-fit line for these standards with the equation of the line.
- Use the equation of the line to estimate the concentration of the unknown sample.
Curve Fitting
Run the simulation below to understand how you can use the standard dilution series to estimate your sample concentrations.
Click on the image above to begin simulation on curve fitting.
Scatterplot Tutorial
Use the tutorial below and watch at 1.25X to plot your own data.