Protein Assay by the Bradford Method Finale

Protein Assay by the Bradford Method

Experiment no. 3Arao

BascoCortesFlores

GochocoMabunay

IntroductionProtein Assays.

Absorbance AssayColorimetric Assay

IntroductionAbsorbance Assay

Spectrophotometric Assay280 nm.

IntroductionColorimetric Assay

Biuret AssayLowry Protein AssayBradford Assay

Colorimetric AssayBiuret Assay

Biuret reagent: alkaline copper sulfateviolet

Lowry Protein AssaySensitive; blue colorColor development is similar to Biuret Assay but

uses a second reagent: Folin- Ciocalteca

Bradford Methodbinding of the dye Coomassie Brilliant Blue G-250 to

proteins

Coomassie Brilliant Blue G-250

IntroductionCoomasie Brilliant Blue G-250

Exists in three forms: cationic(red), neutral (green), and anionic (blue)

Introduction H+ H+Cation ↔ Neutral form ↔ Anion470 nm (red) 650 nm (green) 595 nm (blue)

IntroductionAdvantages of Bradford Method.

Accurate High SensitivityRapidFew interferences by non protein components

Detergent, Triton x-100, and sodium dodecyl sulfate

IntroductionDisadvantage:

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

Materials Unknown protein solutionBradford ReagentBovine Serum Albumin (BSA)

MethodologyPrepare a set of standards as shown in the table below.

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

MethodologySolution/Tube

# 1 2 3 4 5 6 7 8

BSA Stock solution, mL 0 0.2 0.3 0.4 0.5 0.6 0.8 1.0

Distilled water, mL 1.0 0.8 0.7 0.6 0.5 0.4 0.2 0

MethodologyTo these tubes, add 5 mL of Bradford reagent and mix

well.

Solution/Tube # 1 2 3 4 5 6 7 8

BSA Stock

solution, mL 0 0.2 0.3 0.4 0.5 0.6 0.8 1.0

Distilled water,

mL 1.0 0.8 0.7 0.6 0.5 0.4 0.2 0

Bradford reagent,

ml

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

MethodologyFor the unknown protein solution, use 1.0 mL each in two

trials and add 5mL of Bradford reagentZero 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

Bradford AssayResults and Discussion

Team A

Data of Team Asolutions 1 2 3 4 5 6 7 8

BSA Stock

Solution

0 0.2 0.3 0.4 0.5 0.6 0.8 1

Distilled H2O (mL)

1 0.8 0.7 0.6 0.5 0.4 0.2 0

Bradford Reagent

(mL)

5 5 5 5 5 5 5 5

Absorbance at

595 nm(A)

0.001 0.150 0.190 0.227 0.291 0.303 0.404 0.478

Concentration

(µg/mL)

0 40 60 80 100 120 160 200

Group 1Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 21.0 1.0 1.0 1.0

5 5 5 5.354 .440 .291 .336138 180 108 130

Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.363 0.367 0.341 0.341142.66

67145.33

33131.99

97131.99

97

Group2

Group 3Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.287 0.264 0.350 0.346

106 94 134 133.5

Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.333 0.338 0.297 0.295128.9

7131.1

6113.1

9112.3

1

Group 4

Group 5Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.354 0.367 0.259 0.267

153.59 158.96 111.93 115.44

Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.307 0.305 0.340 0.322

117.5 116.7 132.1 124.1

Group 6

0 2 4 6 8 10 12 14 160

0.1

0.2

0.3

0.4

0.5

0.6

f(x) = 0.0315 x + 0.0460384615384614

Concentration (μg/ml)

Abso

rbance (

595 n

m)

Absorbance versus ConcentrationGroup 1

0 50 100 150 200 2500

0.1

0.2

0.3

0.4

0.5

0.6

f(x) = 0.00228163265306123 x + 0.0387448979591836R² = 0.980611734948852

BRADFORD ASSAY

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

Concentration (μg/mL)

Abso

rbance a

t 595nm

(A

)

Absorbance versus ConcentrationGroup 2

Absorbance versus ConcentrationGroup 3

0 50 100 150 200 2500

0.1

0.2

0.3

0.4

0.5

0.6

Absorbance versus ConcentrationGroup 4

0 50 100 150 200 2500

0.1

0.2

0.3

0.4

0.5

0.6

Absorbance versus ConcentrationGroup 5

Absorbance versus ConcentrationGroup 6

BRADFORD ASSAYResults and Discussion

Team B

Absorbance and Concentration of Standard Protein Solution based on the Spectrophotometer

Solution Standard Test Tubes

  1 2 3 4 5 6 7 8

BSA Stock Sol’n (mL)

0 0.2 0.3 0.4 0.5 0.6 0.8 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)

0 40 60 80 100 120 160 200

Group 7 Group 8Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

 .225

 .235

 .335

 .365

 108

 110

 156

 170

Group 9 Group 10Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.197 0.201 0.292 0.293

80 81 122 123

Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

0.284 0.272 0.224 0.214

124 117 91 95

Group 11Unknown 1 Unknown 2

Trial 1 Trial 2 Trial 1 Trial 2

1.0 1.0 1.0 1.0

5 5 5 5

174 166 176 179

0.330 0.310 .337 .348

0 50 100 150 200 2500

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Absorbance

Concentr

ati

on

Absorbance versus ConcentrationGroup 7

Concentration BSA µg/mL

Absorbance versus ConcentrationGroup 8

Absorbance versus ConcentrationGroup 9

Absorbance versus ConcentrationGroup 10

0 50 100 150 200 250

-0.1

0

0.1

0.2

0.3

0.4

0.5

f(x) = 0.00205051020408164 x + 0.0228265306122448R² = 0.982960372486107

Bradford Assay

Absorbance at 595 nm (y)Linear (Absorbance at 595 nm (y))

BSA Concentration ɥg/mL

Abso

rbance a

t 595 n

m (

y)

Absorbance versus ConcentrationGroup 11

0 50 100 150 200 250

-0.1

0

0.1

0.2

0.3

0.4

0.5

Series1Linear (Series1)

Chemical Reaction of Coomasie Dye with Protein

Computation for Concentration

Computation for ConcentrationUsing the Dilution formula: C1V1=C2V2

C2 = Ctesttube#

Test tube #1:(200µg/mL)(0.0mL)=C2(1.0mL)

Ctesttube1= 0µg/mL

Test tube #2:(200µg/mL)(0.2mL)=C2(1.0mL)

Ctesttube2= 40µg/mL

Test tube #3:(200µg/mL)(0.3mL)=C2(1.0mL)

Ctesttube3= 60µg/mL

Test tube #4:(200µg/mL)(0.4mL)=C2(1.0mL)

Ctesttube4= 80µg/mL

Test tube #5:(200µg/mL)(0.5mL)=C2(1.0mL)

Ctesttube2= 100µg/mL

Test tube #6:(200µg/mL)(0.6mL)=C2(1.0mL)

Ctesttube6= 120µg/mL

Test tube #7:(200µg/mL)(0.8mL)=C2(1.0mL)

Ctesttube7= 160µg/mL

Test tube #8:(200µg/mL)(1.0mL)=C2(1.0mL)

Ctesttube2= 200µg/mL

Principle behind Bradford AssayBradford Assay

colorimetric assay for measuring protein concentration in a given solution.

Involves binding of the dye Coomassie Brilliant Blue G-250 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

ConclusionProtein 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.