Heather Elia

BRADFORD PROTEIN ASSAY

Abstract: In this lab we used the Bradford Method to determine the
protein concentrations of 3 unknown values. We measured the
absorbance of various concentrations as well as the unknowns to
determine the protein concentration of each one. We found absorbance
using the spectrophotometer. For our unknown values absorbance
was; unknown #1- 0.264, unknown #2-0.424, and unknown #3-0.141.
Introduction: In this exercise, you learn and apply Bradford assay
(specifically the assay developed by the Bio-Rad Company) to determine
the amount of protein in an unknown solution. This technique will be
consequently used in future experiments to determine the level of soluble
proteins in food items. The Bradford assay is a very useful tool in

determining the concentration of certain proteins. It measures the

amount of protein (any and all proteins). It is based on a dye,
Coomassie Brilliant Blue G250. It binds to basic and aromatic amino
acids. The absorbance maximum shifts from 465 nm (brownish) to 595
nm (blue) when binds to protein. It works on the basis of
spectrophotometry: as the concentration of a solution increases, so
does its absorbance. We are able to plot a known curve (standard) of
absorbance vs. concentration, and then extrapolate our absorbance
values to obtain concentration values for unknowns. This is a
necessary technique to food science, as the ability to determine the
concentrations of certain proteins in foods is sometimes needed.

Materials:
1. Distilled water
2. Spectrophotometer
3. Dye
4. Vortex
5. Cuvette
6. Micropipettor
7. 12 Test tubes
8. Micropipette tips

Methods:
1. Add 20ml of dH2O to Bio-Rad Protein (lyophilized BSA standard;
1.40mg/ml final conc.)
2. Make a series of protein concentration gradients
100, 200, 400, 600, 800, 1000, 1200, 1400 ug/ml
3. Take 50ul of each standard and pipette into 5ml tube, include step 1 of
part to at this time.
4. Add 2.5ml of dye (diluted 1:4!!) and add to each 5 ml tube. Vortex for
20 seconds.

5. Pipette 2ml of std into cuvette

6. Obtain absorbance for each standard using spectrophotometer

PART II- Sample Analysis

1. Pipette 50ul of each sample into 5ml tube. Unknown sample is
different concentrations of FBS.
2. Add 2.5ml of dye (diluted 1:4!!) and vortex for 20 sec
3. Allow samples to sit for 5 minutes
4. Pipette 2ml sample into cuvette
5. Obtain absorbance and protein concentration for your 3 unknowns
For spectrophotometer: Select protein assay, Bradford, change to g/ml.
dilution 10.

Results:
Concentration

BSA

dH2O (adjust to

Absorbanc

1ml)
929 ul
858 ul
716 ul
574 ul
432 ul
280 ul
148 ul
6 ul

e
0.093
0.149
0.260
0.309
0.516
0.628
0.712
0.716

(ug/mL)
(1.40mg/ml)
100
71 ul
200
142 ul
400
284 ul
600
426 ul
800
568 ul
1000
710 ul
1200
852 ul
1400
994 ul
Unknown #1: 0.264 ~ 400 ug/mL
Unknown #2: 0.424 ~ 600-800 ug/mL

Unknown #3: 0.141 ~ 200 ug/mL

Unknown calculated concentrations:

Equation: Y=0.101x-0.318
Unknown #1:

Unknown #2:

Unknown #3:

0.264

0.424

0.141

0.264=0.101x-

0.424=0.101x-

0.141=0.101x-

0.318

0.318

0.318

x= 434

x= 734

x= 154

Discussion:
A common method to prepare a standard curve is to prepare various
known protein concentrations as standards. As long as the volume of
the standard samples and the unknown samples are the same the final
concentration of the unknown is directly calculated from the least
squares line of the standard curve. [1]

According the Lambert-Beer law, an increase in the concentration of a

solution leads to a increase in the absorbance as linear. These data
are used to make the standard curve, plotting concentration on the X
axis, and assay measurement on the Y axis. The same assay is then
performed with samples of unknown concentration. To analyze the
data, one locates the measurement on the Y-axis that corresponds to
the assay measurement of the unknown substance and follows a line
to intersect the standard curve. The corresponding value on the X-axis
is the concentration of substance in the unknown sample. [2]
Trend lines are often used to argue that a particular action or event
that causes observed changes at a point in time. [3] This graph
showed an upward trendline. If the plots are above the standard curve
it is a higher optical density and if they are below this indicated that it
is a lower optical density. The calculated equation for the line was
Y=0.101x-0.318. This was used to determine the concentrations of the
unknown values. According to our results, our unknown #1 matched
up with approximately concentration 400 (434) ug/mL and our
unknown #3 matched up with concentrations 200 (154) ug/mL. Our
unknown #2 didnt match up with any of the values but it falls inbetween the concentrations of 600 ug/mL and 800 (734) ug/mL. This
could be due to human error such as from pipetting, having multiple
pipetters, improper handling or improper vortexing of the solution.
For the most part, all of our absorbencies matched up between the
given values or were relatively close. This pretty much tells us that our
results came out pretty accurate with the standard curve. Human error,
however, could have occurred with the pipetting.
Acknowledgements:
Professor Lisa Lucente
Lab group members

Bibliography:

Lisa Lucentes class slideS ANALYSIS OF PROTEIN METHODS

USED IN PROTEIN DETERMINATION

PDF of Proteins (experiment 5)

Bradford method worksheet

[1]
http://www.mnstate.edu/provost/BradfordProteinAssayProtocol.pd
f

[2] http://en.wikipedia.org/wiki/Standard_curve

[3] http://en.wikipedia.org/wiki/Linear_regression