protein assay by the bradford method finale
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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.