Krzysztof Treder, Treder Lab http://ziemniak-bonin.pl/en/?

option=com_content&view=article&id=77

Micro-Bradford assay for determination of protein concentration

Reagents:
Bradford Reagent (5X concentrate)
100 mg Coomassie Brilliant Blue G-250
47 ml Methanol (100%)
100 ml Phosphoric Acid (85%)
Fill to 200 ml with H2O
Coomassie must be dissolved in the methanol FIRST before the other ingredients are added. Reagent is
stable for at least 1 year but mix well before using. This reagent is sold ready to use by many
companies.

Standard proteins – 10 mg/ml of Bovine serum albumin (BSA) and Bovine gamma-globulin (BGG) or
pure preparations of these proteins in powder, or commercial pre-diluted standards covering the
wanted concentration range.

PBS pH 7.4:
Dilute in 900ml H2O:
NaCl 8.00g
KH2P04 0.20g
Na2H P04 x12 H2O 2.90g
KCl 0.20g
NaN3 (optional) 0.20g
Check pH and if necessary titrate it to 7.4 with 1M HCL or 1M NaOH. Fill to 1 L and filtrate trough 0.45
um filter.
NaN3 is a poison, toxic to human. This is inhibitor of oxidoreductases and prevents microbial growth.
If you need to avoid NaN3, buffer can be divided into 50-100 ml bottles and autoclaved instead. With
NaN3 buffer is stable for 6 months at room temperature, autoclaved – can be stored for 1-2 years if
not opened and kept in dark. Instead of PBS pure phosphate or TRIS buffers can be used.

Method:
For albumin-like proteins prepare stock solution of BSA in 1xPBS – 1mg/1ml. For globulin-like proteins
do the same using BGG (http://www.piercenet.com/product/coomassie-bradford-protein-assay):

- by dilution of commercial stock of 10mg/ml of BSA or BGG

- or by preparing 10mg/ml stock from BSA or BGG powder and next diluting (for example weight
100 mg of BSA/BGG powder and dissolve it 1xPBS in final volume of 10 ml, next take 100 µl of
the stock and add 900 ul of 1xPBS to prepare 1 mg/ml solution). This will lower error in your
stock concentration.

Next prepare protein concentration standards from 1mg/ml BSA or BGG (1000 µg/ml = 1 µg/µl):

standards
10 20 30 40 50 70 80
[µg/ml]
BSA [µl] 10 20 30 40 50 70 80
1 x PBS [µl] 990 980 970 960 950 930 920
 Krzysztof Treder, Treder Lab http://ziemniak-bonin.pl/en/?option=com_content&view=article&id=77

If you want to be very accurate you should prepare 2 separate BSA stocks of 1mg/ml and next 2
separate series of standards.

Dilute your samples in 1xPBS from 2 to 100-fold (for example: 10 and 50-fold)
Dilute your sample buffer the same way as samples.

To microplate wells add:

110 µl of 1 x PBS
50 µl of standard or sample or sample buffer (diluted the same way as sample protein)
40 µl of concentrated Bradford reagent

Mix well and wait at least 5-15 minutes but not longer than 1 hr (protein-dye complexes precipitate
after some time, for fresh homemade Bradford reagent use 5 min). Next read at 595 nm in microplate
reader.

Example of micro-plate organization:

diluted samples (example fold dilutions)

from stock I from sock 2 10 10 10 50 50 50

1 2 3 4 5 6 7 8 9 10 11 12
sample sample sample sample sample sample
A 0 0 0 0 0 0
1 1 1 1 1 1
sample sample sample sample sample sample
B 10 10 10 10 10 10
2 2 2 2 2 2
sample sample sample sample sample sample
C 20 20 20 20 20 20
3 3 3 3 3 3
sample sample sample sample sample sample
D 30 30 30 30 30 30
4 4 4 4 4 4
sample sample sample sample sample sample
E 40 40 40 40 40 40
5 5 5 5 5 5
sample sample sample sample sample sample
F 50 50 50 50 50 50
6 6 6 6 6 6
sample sample sample sample sample sample
G 70 70 70 70 70 70
7 7 7 7 7 7
Buffer Buffer Buffer Buffer Buffer Buffer
H 80 80 80 80 80 80
1 1 1 2 2 2

Prepare a calibration plot by graphing the net A595 values for the standards versus protein
concentration. Determine the protein concentration of the sample by interpolation from the plot.

Data points on the plot can be fit to linear regression and unknown sample concentration can be
determined from equation. Simple linear regression can be performed in Excel or LibreOffice. More
accurate linear and non-linear regression can be performed in GraphPad Prizm or in SigmaPlot.
 Krzysztof Treder, Treder Lab http://ziemniak-bonin.pl/en/?option=com_content&view=article&id=77

Example of calculations using Excel:

1. Calculate average OD for protein standards
2. Plot the concentration (Y) as a function of OD (X).
3. Fit the points using linear regression, check option to show regression equation.
4. Use the equation to calculate concentration of unknown protein samples using their OD as X in
the equation.

Optical dencity Standard OD minus Standard

(OD=595 nm) deviation background Protein

X1 X2 X3 Average SD 595 nm ug/ml

0,138 0,138 0,117 0,131 0,000 0
0,327 0,333 0,317 0,326 0,008 0,195 10
0,502 0,488 0,489 0,493 0,008 0,362 25
0,694 0,695 0,708 0,699 0,008 0,568 40
0,831 0,869 0,874 0,858 0,024 0,727 50
1,139 1,161 1,166 1,155 0,014 1,024 70
1,525 1,525 1,396 1,482 0,074 1,351 100

In this example unknown protein concentration in µg/ml (y) = 73,58xOD595-2,291