Lab. 2. PROTEIN ASSAY

The purpose of this laboratory technique is to determine the amount of protein present in a sample. The sample might be blood, a tissue homogenate, or proteins secreted from bacteria. Or the sample could be a sample of food or beverage, to verify the protein content claimed on the label. In this case we are analyzing samples of, albumin, specifically Bovine Serum Albumin (BSA). BSA is the most abundant protein in serum (blood, minus red and white cells). Assignment: What are the functions of this protein? What is the size of this protein – molecular weight, number of amino acids? Other interesting facts about albumin? Also, obtain this information for actin and myosin proteins. Be prepared to report your findings next class.

We will be using the BioRad Quick Start (Bradford) reagent. This reagent contains Coomassie Blue, a dye the specifically binds to protein molecules and changes color. The same dye is used to stain proteins in gels. The intensity of the color is related to the amount of protein. Thus you can mix known amounts of protein (e.g. BSA) with the dye reagent and measure the intensity of the color in a Spectrophotometer and then compare the intensity from an unknown sample to estimate its protein concentration. http://www.piercenet.com/products/browse.cfm?fldID=02020105

Materials. Each student needs 4 glass tubes ~ 3 ml of BioRad Quick Start (Bradford) reagent or 10 ml for a team of 4.~100 ul of the Bovine Serum Albumin (BSA) standard solution, 1 ug/10 ul (=1 mg/ 10 ml).Distilled Water in a 15 ml tube.One, 1 ml plastic cuvetteEach team needs a tube rack, and a spectrophotometer.

Methods. Each student should make a table (Table 1) in their note book that shows the volumes of BSA, water and BioRad Reagent that will be added to each tube and indicate the amount of protein (ug in each tube). Tubes should be prepared for each standard amount of BSA: 0, 10, 20 and 50 ul plus distilled water to bring the volume in each tube up to 500 ul. Also, prepare tubes two different amounts of your unknown protein sample plus water to 500 ul. Date the page. If these sample volumes do not yield Absorbance values within the range of the BSA standards you should try different amounts.

Once all of the tubes, standards and unknowns, have been prepared add 500 ul of the BioRad reagent to each tube. After this addition the total volume in each tube, standards and unknowns, should be 1000 ul. As you add the dye mix each tube several times so that the color is uniform.

Set up the spectrophotometer to read the light Absorbance of each sample. Calibrate the spectrophotometer with dH2O. Make sure that the cuvette is oriented so that the light path passes through the clear windows of the cuvette. Set the wavelength to 595 nm. Pour out the water, pour in your standard “0” into the cuvette and write down the Absorbance reading in the Table I your notebook. Pour the “0” standard back into its tube and repeat the process with each of the other standards and unknowns, recording the readings in Table 1.

Results as they should appear in your note book:

A. Table I showing the ul water, ul BSA, BSA, ul unkown, ug protein in unknown and the Absorbance readings for everyone in your group.

B. Plot all of the standard values for your group (not averages) with the ug BSA values on the x-axis and the Absorbance (A695) on the y-axis. Draw a line or curve that best fits the points. If your standard points do not describe a clear line or curve repeat the Methods. If the A695 readings for your unknowns are greater than your standard readings repeat the unknown with a smaller amount of sample or with a diluted sample. Or if the readings are low use a greater amount of the unknown sample. Keep exact notes on what volumes you pipette to make the dilutions.

• Challenge question: what is the molar concentration (M – moles/liter) of BSA in the standard solution and how many water molecules are there per BSA molecule in that solution?

C. Concluding statements: Describe your observations and uncertainties. Give an estimate for the ug/ul protein in your unknown. Suggest what you could do to improve your measurements and results.

Show this to your lab instructor before leaving today.