Week #1 Laboratory Excercise: Determination of A Standard Curve for Protein Concentration.
The purpose of this exercise is to allow you to become familiar with some of the laboratory equipment you will be using throughout this course. You will test your skill in using the P-20 and P-200 micropipettes by constructing a standard curve for determination of the concentration of an unknown protein. If you use the pipettes correctly, the curve that you generate by plotting absorbance vs concentration of an unknown protein should be a straight line. The value that you determine for the concentration of your unknown protein sample should be within 10% of the actual value that is known to your instructors. This exercise will not be graded and you will not be required to hand in a written report. However, the experience you will gain this week will be essential in order to complete Lab Exercise #1 with precision and within the 4 hr time period.
Protein concentrations will be determined using the method of Bradford. The Bradford procedure is a dye-binding assay based on the differential color change of Coomassie blue G dye as it binds to protein. In both research and clinical applications, this assay has replaced earlier procedures for protein measurement (i.e. the Lowery method) because of its simplicity, the stability of the color change, and the lack of interference from non-protein components.
The Bradford assay is based on the observation that the absorbance maximum for an acidic solution of the dye shifts from 465 nm to 595 nm when binding to protein occurs. Beer's Law (see Lab #1) may be applied for accurate quantitation of protein in solution by selecting an appropriate ratio of dye volume to sample concentration. Within a range of 5-100 ug of protein, a linear relationship exists between protein concentration and the increase in absorbance of the dye solution at 595 nm. Over a broader range of protein concentration, the dye binding method gives an accurate, but not entirely linear response.
1. The instructor will provide a solution of a purified protein, bovine serum albumin (BSA), at a known concentration (10 mg/ml).
Make a series of dilutions of the BSA standard solution (10 mg/ml) into 1.5 ml microcentrifuge tubes to give final concentrations of 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml of protein. Make 0.1 ml (100 ml) of each dilution. As a diluent, use filtered water.
2. Place 20 ml of each dilution into individual microcentrifuge tubes and make up an additional tube containing 20 ml of water for use as a reagent blank. Add 1 ml of diluted Bradford dye reagent to each tube, close the tube, and vortex to mix thoroughly. After at least 5 min, but before 1 hr, transfer 1 ml to a plastic cuvette. Use the reagent blank to zero the spectrophotometer at 595 nm, then read the absorbance of each sample at 595 nm. Record the absorbance of each sample in your notebook.
3. Plot, either on graph paper or on the computer using the program Excel, the absorbance of each sample vs. the protein concentration in mg. Make a second graph in which the absorbance of each sample is plotted vs. the AMOUNT of protein contained in each assay. The line that best runs through the points on each graph represents the standard curve. Note that the Bradford assay is subject to variation from day-to-day, and from one batch of dye reagent to another. Therefore, it is necessary to construct a new standard curve each day, or when a different bottle of reagent is used.
If your group’s line is not straight, then you will have to repeat the procedure until your results are reproducible.
4. Obtain from your instructor a sample of protein at an "unknown" concentration. Using the Bradford assay, and your standard curve, determine the following values: