Part A: The protein assay
Introduction:

It is often necessary to determine the concentration of protein in dilute solutions. A sensitive yet simple colorimetric assay that has gained wide usage was developed by Bradford. The method involves mixing a dye, called Coomassie Brilliant Blue, directly with a protein solution. The dye forms a complex with protein. When the dye binds to protein it changes color, which means that it absorbs light at a different wavelength. The absorption maximum is shifted from 465 to 595 nm. With a spectrophotometer it is possible to quantify color by measuring the amount of light that the solution absorbs. At 595 nm absorption is linear over a specific range of protein.
A standard curve provides a reference for measuring the amount of protein in a solution of unknown concentration. It is constructed by measuring the absorption of several known concentrations of protein in the range of 0-2000 ug. Then, when a solution of unknown concentration is measured, its absorbance at 595 nm can be compared to the standard curve. The trick is that the concentration of the unknown must be in the range of the standard curve, that is somewhere between 0-2000 ug. The dilemma, however, is that the concentration of the unknown is not known.
A useful strategy for dealing with unknowns is to prepare serial dilutions. Because the linear portion of the standard curve extends over a full order of magnitude of concentration, a fail-safe approach for a serial dilution of the unknown is one based on the order-of- magnitude dilutions.
This exercise involves two parts. First, a standard curve will be constructed using a solution of known concentration of bovine serum albumin (BSA). Then, using the strategy of serial dilution, the concentration of a mystery solution of BSA (labeled X, Y or Z) will be determined.

Procedure:
1. The standard curve
Obtain a solution of protein whose concentration is known. Bovine serum albumin (BSA) has been prepared at a concentration of 2 mg/ml.
Label a set of test tubes from 1-7 using a black Sharpie marker or pen on tape. Following the chart below fill the tubes sequentially with BSA, waster and Bradford reagent. Bradford reagent contains a dye, Coomassie blue, which binds to protein. The dye/protein complex produces a blue color whose absorbance is directly proportional to the protein concentration.

Volume of BSA to Add Volume of Diluent to Add Final BSA Concentration Tube

300 ul stock 0 ul 2,000 ug/ml
375 ul stock 125 ul A
325 ul stock 325 ul B
175 ul of A 175 ul C
325 ul of B 325 ul D
325 ul of D 325 ul E
325 ul of E 325 ul F

NOTE: It is important to mix the tubes rapidly and thoroughly immediately after the dye is added, one tube at a time. Because the Bradford reagent contains phosphoric acid, avoid contact with the mouth or skin.

Enter the amount of protein each tube contains in the column headed "ug/tube."
Full color development should occur in 5 minutes. Assay the tubes with the spectrophotometer within 1 hour. Use the tube #1, the BLANK, to set the absorbance at 595 nm to zero. Measure the A595 of each of the other tubes. Write the values in the chart above.
Plot your data on graph paper, ug protein on the X axis and A595 on the Y axis. Show your graph to the instructor. If a linear graph is not obtained, repeat the assay being more careful with pipetting.

2. Assay of the unknown
In this portion of the laboratory you will determine the concentration of an unknown solution of BSA. Dealing with unknowns, determining as much information about them as possible, is what makes science so much fun. However, the enjoyment factor is nearly canceled out by the frustration level if the investigation is not carried out in an orderly fashion. For example, the way to ascertain the concentration of BSA in the unknown is to compare its absorbance to the standard curve created in the first part of this exercise. You might guess that a five-fold dilution might do the trick. However, if the A595 does not fit the curve, it will be necessary to dilute the sample again. If the absorbance reading still does not fit, you would have to try yet another dilution. This "shot in the dark" technique not only takes a lot of time but uses up quite a bit of sample and provides plenty of opportunity for error.

The most efficient way to determine the concentration of an unknown is by using the strategy of the serial dilution. By serially diluting the unknown by an order of magnitude (10-fold) each time, you will be guaranteed to have one tube with an absorbance value that fits on the standard curve. Once the concentration of unknown is determined for a particular tube, it is only a matter of a simple "back calculation" to determine the amount of protein in the original sample.

Label a set of test tubes 1-4 and pipette 0.9 ml of water into each tube. To the first tube add 0.1 ml of unknown for a total of 1.0 ml. Tube 1 now contains a 10-fold dilution of the unknown or an order of magnitude less protein than the original tube. Mix the tube gently by pipetting up and down; try to avoid creating bubbles. This action also rinses the walls of the pipette so that any solution remaining in the pipette will be of the same protein concentration as the tube. Why is this important?

Transfer 0.1 ml from tube 1 to tube 2; mix gently. The second tube now contains a 10- fold dilution of the protein in the first tube, or 2 orders of magnitude (10 x 10) less protein than the original (Co x 10-2). Get the idea?

Now transfer 0.1 ml from tube 2 to tube 3, mix gently. Transfer 0.1 ml from tube 3 to tube 4. How many orders of magnitude less than the original do you have in this last tube? Express it as a fraction. Express it also as an exponent. What volume of solution would you have ended up with if you had made this dilution directly from the original sample? In other words, starting with 0.1 ml of unknown, how much diluent would be necessary to achieve this same degree of dilution without using the serial dilution technique?

Remove 0.1 ml from tube 4 and discard it (i.e. blow it into the sink). What is the reason for this?
Add 0.1 ml of water and 5 ml of the Bradford Reagent to each tube and measure the A595. Use the tube with the "best fit" to the standard curve for the back calculation.

For example, if the amount of protein in tube 2 is 35 ug (in a 1 ml volume), then the concentration in the original tube is
10 x 10 x 35 = 3500 ug/ml = 3.5 mg/ml