Experiment no.

3 Arao Basco Cortes Flores Gochoco Mabunay

Introduction Protein Assays.

Absorbance Assay

Colorimetric Assay

Introduction
Absorbance Assay
Spectrophotometric Assay

280 nm.

Introduction
Colorimetric Assay
Biuret Assay Lowry Protein Assay Bradford Assay

Colorimetric Assay
Biuret Assay Biuret reagent: alkaline copper sulfate violet

Lowry Protein Assay Sensitive; blue color Color development is similar to Biuret Assay but uses a second reagent: Folin- Ciocalteca

Bradford Method
binding of the dye Coomassie Brilliant Blue G-250 to

proteins

Coomassie Brilliant Blue G-250

Introduction
Coomasie Brilliant Blue G-250
Exists in three forms:

cationic(red), neutral (green), and anionic (blue)

Introduction
H+ H+ Cation Neutral form Anion 470 nm (red) 650 nm (green) 595 nm (blue)

Introduction
Advantages of Bradford Method. Accurate High Sensitivity Rapid Few interferences by non protein components

Detergent, Triton x-100, and sodium dodecyl sulfate

Introduction
Disadvantage:

The dye reagent reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues.

Materials
Unknown protein solution
Bradford Reagent Bovine Serum Albumin (BSA)

Methodology
Prepare a set of standards as shown in the table below. Also

prepare a reagent blank consisting of 1.0mL of distilled water (tube #1)

Methodology
Solution/Tube # BSA Stock solution, mL Distilled water, mL 1 2 3 4 5 6 7 8 0 0.2 0.3 0.4 0.5 0.6 0.8 1.0

1.0

0.8

0.7

0.6

0.5

0.4

0.2

Methodology
To these tubes, add 5 mL of Bradford reagent and mix

well.
Solution/ Tube # BSA Stock solution, mL Distilled water, mL Bradford reagent, ml 1 2 3 4 5 6 7 8

0.2

0.3

0.4

0.5

0.6

0.8

1.0

1.0

0.8

0.7

0.6

0.5

0.4

0.2

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5. 0

Methodology
For the unknown protein solution, use 1.0 mL each in two

trials and add 5mL of Bradford reagent Zero the spectrophometer using the reagent blank. After 5 min, but before one hour, read the absorbance of the standards and the unknown protein solution at 595nm (A595) against a reagent blank. Draw the standard curve plot by plotting A595 versus the concentration of BSA. Calculate the concentration of the protein solution by comparison with the standard curve for BSA

Results and Discussion Team A

Data of Team A
solutions BSA Stock Solution Distilled H2O (mL) Bradford Reagent (mL) Absorba nce at 595 nm (A) Concentr ation (g/mL)

1 0 1 5 0.001

2 0.2 0.8 5 0.150

3 0.3 0.7 5 0.190

4 0.4 0.6 5 0.227

5 0.5 0.5 5 0.291

6 0.6 0.4 5 0.303

7 0.8 0.2 5 0.404

8 1 0 5 0.478

40

60

80

100

120

160

200

Group 1
Unknown 1 Trial 1 Trial 2 1.0 1.0 Unknown 2 Trial 1 Trial 2 1.0 1.0

Group2
Unknown 1 Trial 1 1.0 5 0.363 Trial 2 1.0 5 0.367 Unknown 2 Trial 1 1.0 5 0.341 Trial 2 1.0 5 0.341

5 .354 138

5 .440 180

5 .291 108

5 .336 130

142.6667 145.3333 131.9997 131.9997

Group 3
Unknown 1 Trial 1 1.0 5 0.287 Trial 2 1.0 5 0.264 Unknown 2 Trial 1 1.0 5 0.350 Trial 2 1.0 5 0.346

Group 4
Unknown 1 Unknown 2

Trial 1
1.0 5 0.333 128.97

Trial 2
1.0 5 0.338 131.16

Trial 1
1.0 5 0.297 113.19

Trial 2
1.0 5 0.295 112.31

106

94

134

133.5

Group 5
Unknown 1 Trial 1 1.0 Trial 2 1.0 Unknown 2 Trial 1 1.0 Trial 2 1.0

Group 6
Unknown 1 Trial 1 1.0 Trial 2 1.0 Unknown 2 Trial 1 1.0 Trial 2 1.0

5
0.354

5
0.367

5
0.259

5
0.267

5
0.307

5
0.305

5
0.340

5
0.322

153.59

158.96

111.93

115.44

117.5

116.7

132.1

124.1

Absorbance versus Concentration Group 1

0.6 y = 0.0315x + 0.046 0.5

Absorbance ( 595 nm)

0.4

0.3

0.2

0.1

8
Concentration (g/ml)

10

12

14

16

Absorbance versus Concentration Group 2

BRADFORD ASSAY
0.6

0.5
Absorbance at 595nm (A)

y = 0.0023x + 0.0387 R = 0.9806

0.4

0.3

Absorbance at 595 nm (A) Linear (Absorbance at 595 nm (A))

0.2

0.1

0 0 50 100 150 200 250 Concentration (g/mL)

Absorbance versus Concentration Group 3

Absorbance versus Concentration Group 4

0.6

0.5

0.4

0.3

0.2

0.1

0 0 50 100 150 200 250

Absorbance versus Concentration Group 5

0.6

0.5

0.4

0.3

0.2

0.1

0 0 50 100 150 200 250

Absorbance versus Concentration Group 6

Results and Discussion Team B

Absorbance and Concentration of Standard Protein Solution based on the Spectrophotometer

Solution Standard Test Tubes 1 BSA Stock Soln (mL) 0 2 0.2 3 0.3 4 0.4 5 0.5 6 0.6 7 0.8 8 1.0

Distilled H2O (mL)

1.0

0.8

0.7

0.6

0.5

0.4

0.2

0.0

Bradford Reagent (mL) 5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

Absorbance (595nm)

0.001

0.108

0.146

0.210

0.246

0.277

0.362

0.429

Concentration (mg/mL)

40

60

80

100

120

160

200

Group 7
Unknown 1 Trial 1 1.0 Trial 2 1.0 Unknown 2 Trial 1 1.0 Trial 2 1.0

Group 8
Unknown 1 Trial 1 1.0 Trial 2 1.0 Unknown 2 Trial 1 1.0 Trial 2 1.0

5 5 5 5 5 .225

5 .235

5 .335

5 .365

108

110

156

170

Group 9

Unknown 1 Trial 1 1.0 Trial 2 1.0

Unknown 2 Trial 1 1.0 Trial 2 1.0

Group 10

Unknown 1 Trial 2 1.0

Unknown 2 Trial 1 1.0 Trial 2 1.0

Trial 1 1.0

5
0.197 80

5
0.201 81

5
0.292 122

5
0.293 123

5
0.284 124

5
0.272 117

5
0.224 91

5
0.214 95

Group 11
Trial 1 1.0

Unknown 1 Trial 2 1.0

Unknown 2 Trial 1 1.0 Trial 2 1.0

5 174 0.330

5 166 0.310

5 176 .337

5 179 .348

Absorbance versus Concentration

Group 7
0.5 0.45 0.4 0.35 Concentration 0.3 0.25

0.2
0.15 0.1 0.05 0 0 50 100 Absorbance 150 200 250

Absorbance versus Concentration

Group 8

Concentration BSA g/mL

Absorbance versus Concentration

Group 9

Absorbance versus Concentration

Group 10

Bradford Assay
0.5 0.45 0.4 Absorbance at 595 nm (y) 0.35 0.3 0.25 Absorbance at 595 nm (y) Linear (Absorbance at 595 nm (y))

y = 0.0021x + 0.0228 R = 0.983

0.2
0.15 0.1 0.05 0 -0.05 0 50 100 150 BSA Concentration g/mL 200 250

Absorbance versus Concentration

Group 11
0.5 0.45 0.4 0.35 0.3

0.25
0.2

Series1 Linear (Series1)

0.15
0.1

0.05
0 0 50 100 150 200 250

-0.05

Chemical Reaction of Coomasie Dye with Protein

Computation for Concentration

Computation for Concentration

Using the Dilution formula: C1V1=C2V2 C2 = Ctesttube# Test tube #1: (200g/mL)(0.0mL)=C2(1.0mL) Ctesttube1= 0g/mL Test tube #2: (200g/mL)(0.2mL)=C2(1.0mL) Ctesttube2= 40g/mL Test tube #3: (200g/mL)(0.3mL)=C2(1.0mL) Ctesttube3= 60g/mL Test tube #4: (200g/mL)(0.4mL)=C2(1.0mL) Ctesttube4= 80g/mL Test tube #5: (200g/mL)(0.5mL)=C2(1.0mL) Ctesttube2= 100g/mL Test tube #6: (200g/mL)(0.6mL)=C2(1.0mL) Ctesttube6= 120g/mL Test tube #7: (200g/mL)(0.8mL)=C2(1.0mL) Ctesttube7= 160g/mL Test tube #8: (200g/mL)(1.0mL)=C2(1.0mL) Ctesttube2= 200g/mL

Principle behind Bradford Assay

Bradford Assay colorimetric assay for measuring protein concentration in a given solution. Involves binding of the dye Coomassie Brilliant Blue G250 to protein in acidic solution Results in spectral shift from reddish brown form of the dye (Absorbance maximum at 465nm)to the blue form of the dye (Absorbance maximum at 610nm)

Why is bovine serum albumin (BSA) used as a standard?

BSA gives a color yield similar to that of the protein being assayed Best relative standard to use for Bradford method

Why is absorbance read at 595, not in any wavelength between 575nm and 615nm?
At these two extremes, there is a loss of about 10% in

the measured amount of color compared to that obtained at 595nm. Bound molecules are most readily detected at 595nm

Conclusion

Protein assay by the Bradford method is in fact one of the many ways to asses the amount of protein in a sample.

An advantage using the Bradford method: good accuracy and convenient A disadvantage : linear over a short range, High concentration detergents can interfere.

Applications: Important not only to chemists.