Chapter 3:

Procedure 3.1
The Bradford Microassay

Background

The basis of the Bradford assay is the observation that certain amino acids found in most proteins selectively bind the dye, Coomassie Brilliant Blue, also called Bradford’s reagent. Under acidic conditions when the dye binds these amino acids, it changes color from reddish-brown to blue. The more of these amino acids there are, the more color change in the solution. This change can be quantitated using a spectrophotometer at 595 nm. We will be using the “Bio-Rad Microassay,” a version of this assay designed to measure 25 µg or less of protein.

For a standard curve, it is necessary to measure the absorbance of “standards” of known concentration, plot the absorbance of the standards, and derive the values for our unknown samples graphically from the values of our standards. The absorbance values of the standards may be graphed in two different ways, either relative to their protein concentration (µg/mL) or to the amount of protein (µg or mg). For these assays, the protein standard will be bovine serum albumin (BSA), a stable, inexpensive protein used for a variety of purposes.

Procedure

These assays are routinely performed using disposable glass tubes. After standards and unknown samples have been prepared and/or diluted, 0.2 ml of dye is added to 0.8 ml of each of the standards and samples. After a short wait to make sure all the dye has bound the protein, the absorbance of the sample is read using a spectrophotometer .

1. Make A Flow Chart. Read through the entire procedure here, and consult the technical literature provided by the manufacturer of the dye (Bio-Rad). Save the flow chart you have prepared because you will be doing this assay many times throughout the purification project.
2. Turn On The Spec. Turn on the spectrophotometer and set the wavelength to 595 nm.
3. Prepare The Standards And Blank. Starting with a BSA stock solution of 1 mg/ml, make a series of dilutions so that the final concentrations range from 1 to 25 µg/ml. (You will need 2 mls of each solution in order to have duplicates.) These are your standards. Table 3.1 is designed to help you with your dilutions. The first line of the table has been filled out for you. Use this as a guide to complete the table. Remember that pipetting 1 µl or less is not very accurate.
4. Pipette 0.8 ml of each standard into a separate tube for assay.
5. In addition, prepare a control tube with 0.8 ml of buffer or water (depending on the diluent for the standards) that will serve as your blank for the spectrophotometer. (If your protein sample of unknown concentration is in a buffer, that buffer will be your blank.)

Table 3.1 BSA DILUTION TABLE FOR STANDARD CURVE
Tube	Final BSA Concentration	Volume of Stock Solution needed In µl	for Volume of H20 µl
1	1.0 ug/ml	2	1998
2	5.0 ug/ml
3	10.0 ug/ml
4	20.0 ug/ml
5	25.0 ug/ml
6(Blk)	0	0	1000
* To determine the amount of stock you will need to make the desired final protein concentrations, use the C1V1 = C2V2 equation, where C1 and C2 are the concentrations of the stock and the diluted sample respectively and V1 and V2 are the volumes of the stock and the diluted sample.



Sample Dilution Calculation:

C₁V₁ = C₂V₂
C₁ = stock concentration of 1 mg/mL or 1000 µg/mL
V₁ = the unknown (volume of stock needed)
C₂ = concentration of BSA standard we are trying to make, 1 µg/ml
V₂ = volume of the standard we are trying to make, 2 mls

1000 µg/ml x V₁ = 1 µg/mL x 2 mL
Solving for V₁ we get an answer of 0.002 mL or 2 µl


6. Prepare The Unknown Samples. In order for the assay to be valid, the unknown sample must be in the concentration range of the standards you have prepared, 1-25 µg/mL. Since you have no idea how much protein there is in the unknown sample, you should prepare several dilutions. What should you use as your diluent? Bear in mind that if none of those dilutions lie within the linear range of the assay, you will need to repeat the entire assay, standards and all. (Why do you have to repeat the ENTIRE assay?)
7. Conduct A Check. At this point you should have 5 standards, a blank (negative control) and several different dilutions of your ß-galactosidase sample. All standards, samples and the blank should have a volume of 0.8 ml. Hold the rack of tubes up to your eye. If the volumes do not appear to be the same, do not proceed. If the volumes are not accurate, the assay will not be accurate and you will be wasting reagents and time if you continue.
8. Add Dye. Add 0.2 ml of Bio-Rad dye to each of the assay tubes. Mix the tubes gently but thoroughly after the addition of the dye: either vortex GENTLY on a low power setting or mix gently by flicking. Avoid foaming!
9. Check For Color. After 5-15 minutes, check to see that color is developing. Solutions without protein should still be reddish brown. Solutions with protein should turn blue: the more concentrated the protein, the more blue the solution should be. You should be able to see the progression of the color in the standard tubes. If not, something is wrong.
10. Read And Record The Samples. After a period of 10 minutes to 1 hour, read the absorbance of each tube at 595 nm on the DU64 Spectrophotometer using plastic cuvettes. The spectrophotometer should be calibrated using the blank (negative control step 5). Then start with the most dilute solution and progress to the most concentrated. Be careful to thoroughly remove the solution from the cuvette each time after reading its absorbance. If you leave some solution in the cuvette, it will dilute the next standard and your curve will not be accurate. Save each solution by putting it back into its tube in case you need to read it again. Record your sample absorbance values in Table 3.1.
11. Prepare The Standard Curve. Generate your standard curve by graphing the absorbance readings of your diluted BSA standards as a function of the protein concentration. The x-axis should be labeled “BSA (µg/µl)” and the y-axis “A₅₉₅”. See Figure 3.1 below.
12. Determine The Sample Values. Figure out the concentration of protein in your unknown(s) by using your standard curve . Record the results in your data table.
13. Do Calculations. Figure out the concentration of protein in the original undiluted sample. (See the sample calculation below.)

Figure 3.1. Sample Bradford Assay Standard Curve.

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

Jeanette Mowery
jmowery@matcmadison.edu
(608) 243-4307