BRADFORD PROTEIN DETERMINATION ASSAY

The purpose of this assay is to determine the amount of protein in a solution. This is often used to standardize 
enzyme assays so that the activities can be expressed as & activity per mg protein ( see your handout)

The process is as follows:

 Get 1 eppendorf tube with BSA (bovine serum albumin-a protein)(10 mg/ml) out of freezer (-20oC).It contains
 100 microliters of BSA. Add 900 microliters of dH2O (dH2O = distilled water) which gives a final stock 
concentration of 1 mg/ml.

Construction of a standard curve:
 

1. 	Set up 6 test tubes labeled in some way to reflect the differences described below:	


2. 	Arrange tubes for the standard curve as follows:
	
		dH2O (microliters)		100	 90	 80	 60	 30	  0
		BSA (microliters)		  0	 10	 20	 40	 70	 100
	 
Run duplicate samples for tubes having 10 and 20 microliters of BSA
	

3. 	Add 5 ml of Bradford reagent - the reagent is stored in the glass container with the pipette apparatus on its top.

4. 	Stir or vortex (using the vibrating mixing device) each tube vigorously. 
	In the standard curve, if you are mixing with a stir stick you may use the same
	one, as long as you start from the lower concentrations and work up to the higher ones. In unknown samples, you
 	may use the same stick only for a single sample. 

5. 	Incubate at room temperature for 2 or more minutes. Color is stable for 1 hour 
	(You may choose any time length under 1 hour, but be consistent across all samples).

6. 	Read the absorbance values in the spectrophotometer at 595 nm. The instrument should be blanked with
 	the sample that has no  protein

7. 	Plot your absorbance values against BSA concenration and calculate the regression coefficient. The more accurate you have pipetted, the closer the value is to 1.0. Normal values are 0.950-0.999.

 	From the standard curve it is possible to calculate the values for unknown samples	

8. 	Determination of unknown values:

	a. Prepare the tubes for a standard curve as described above

b. Make up an additional set of tubes for your unknown samples and add a sample   
    of each of the individual unknowns to a separate tube. It is critical that the 
    absorbance of the samples fall within the values of your standard curve so you 
    may have to use several different volumes per sample unit you have some idea
    of where the absorbances lie. If the samples are very concentrated, they may  
    have to be diluted.  If you do dilute them, you must keep track of the dilution     
    factor so you can correct for it in your final calculations. Remember, each one
    of your unknown samples must have a final volume of 100 microliters before 
    you add the color reagent (volumes used in your standard  curve)for everything 
    to come out correctly.
   

c. Add the 5 ml of reagent to both standard curve tubes and unknowns to develop           
    the color and proceed as described above (Steps 4-7). 

d. Read the standards and then the unknowns. From the linear regression of the   
     standards you can determine the amount of protein in the unknown samples ( 
     caulating an unknown y-value from a known x) Finally, multiply that number
     by the dilution factor if required if you have diluted the protein. To calculate
     the value in mg protein/ml of sample protein you use:

			

	micrograms protein from standard curve x [1000/volume (in microliters) added   
        from the unknown sample]