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Spectrophotometric

Protein Assays
Principles behind dye-based
absorbance and colored complex
measurements for protein
quantification.

OBJECTIVES

Compare different spectrophotometric


BRADFORD
protein assays in terms of accuracy and
precision in protein quantification. LOWRY

Determine the advantages and


disadvantages in biological samples. BIURET
INTRODUCTION
Proteins are large biomolecules consisted of one or
more long chains of amino acid residues. Due to their
function and role in various biological processes,
analysis of these molecules is important.

Quantitative analysis of proteins precedes isolation,


purification, and characterization, making it an
important field in biochemistry.
Spectrophotometry is a method of
determining the amount of light absorbed
(or transmitted) by a chemical substance
over a certain range of wavelength.

The device used is spectrophotometer.


The intensity of light measured is used to determine
the concentration of the substance.
A=bc

The Beer-Lamberts Law is used to relate


absorbance with concentration. Based on
the equation, concentration is directly
proportional to absorbance.
EXPERIMENTAL
Label test tubes
with unknown 1 and
75 mL
2. NaOH
10% (w/v)

Pipette water ,
ovalbumin as indicated 125 mL
BIURET CuSO45H2O
Add Biuret reagent 0.3% (w/v)
and mix thoroughly.
KNaC4H4O64H2O
1.2% (w/v)
Incubate.
*diluted to 250 mL
Measure absorbance,
plot concentration vs.
absorbance.
25 mg
Label test tubes
Coomassie Blue with unknown 1 and
G-250 2.

12.5 mL
Pipette water ,
C2H5OH
95%
BRAD ovalbumin as indicated

25 mL
FORD Add Bradford reagent
and mix thoroughly.
H3PO4
85% (14.7 M)
Incubate.

*diluted with distilled water


to 250 mL and filtered thrice Measure absorbance,
plot concentration vs.
absorbance.
A
Label test tubes CuSO45H2O
with unknown 1 and 1% (w/v)
2.
KNaC4H4O64H2O
Pipette water , 2% (w/v)
ovalbumin as indicated *diluted to 50 mL

Add Lowry reagent LOWRY B


and mix thoroughly.
Na2CO3
Incubate. 2.0% (w/v)

Diluted Folin- NaOH


Ciocalteu phenol,
0.4% (w/v)
let stand
*diluted to 50 mL
Measure absorbance,
plot concentration vs. 50 mL A + 1 mL B
absorbance.
RESULTS &
DISCUSSION
BIURET Table 1. Results from biuret protein assay.

Absorbance at 540 nm Protein


Test Tube Average concentration
Replicate 1 Replicate 2 (mg/mL)
1 0.044 0.043 0.044 0
2 0.117 0.113 0.115 0.333
3 0.208 0.196 0.202 0.667
4 0.308 0.312 0.310 1.00
5 0.348 0.365 0.357 1.33
Unknown 1 0.064 0.084 0.074 0.133
Unknown 2 0.225 0.232 0.229 0.761

RE RE r2 m b
-76.25% -84.78% 0.956 0.232 0.048
Cu2+
complex
The biuret test is used
to detect the presence
of peptide bonds based
on the formation of
copper (II) ion complex
in alkaline solution.
BRAD
Table 1. Results from Bradford protein assay.
FORD
Absorbance at 540 nm Protein
Test Tube Average concentration
Replicate 1 Replicate 2 (mg/mL)
1 - - - 0
2 0.072 0.116 0.094 1.82 x 10-3
3 0.150 0.210 0.180 3.64 x 10-3
4 0.285 0.282 0.284 5.45 x 10-3
5 0.396 0.460 0.428 7.27 x 10-3
6 0.481 0.489 0.485 9.09 x 10-3
Unknown 1 1.156 1.141 1.149 0.0205
Unknown 2 1.976 1.993 1.985 0.0353

RE RE r2 m b
-96.29% -99.28% 0.987 56.67 -0.01496
The Bradford protein assay is
based on the absorbance shift
of the Coomassie Blue G-250
dye. The solution is allowed to
absorb at 465 nm with the use
of the dye in acidic solution
(reddish brown). It then shifts
to 595 nm (blue) when the
negatively charged dye binds
with the positively charged
protein.
Coomassie brilliant blue is a
triphenylmethane dye used to
stain proteins in analytical
chemistry. G-250 has two
additional methyl group.
LOWRY Table 1. Results from Lowry protein assay.

Absorbance at 540 nm Protein


Test Tube Average concentration
Replicate 1 Replicate 2 (mg/mL)
1 - - - 0
2 -0.009 0.001 -4 x 10-3 2.86 x 10-3
3 0.011 -0.003 4 x 10-3 5.71 x 10-3
4 0.045 0.018 0.032 8.57 x 10-3
5 0.035 0.041 0.038 0.0114
6 0.034 0.043 0.039 0.0143
Unknown 1 0.080 0.088 0.084 9.70 x 10-3
Unknown 2 0.459 0.589 0.524 0.0431

RE RE r2 m b
-95.82% -97.44% 0.874 4.20 -0.0142
Biuret The Lowry protein assay
reduction
is an amplified version
of the biuret test by
subsequent reduction
of Folin-Ciocalteu
reagent that gives the
blue color.
Folin-Ciocalteu measures
the total reducing
capacity of a sample. It
also reacts to some
nitrogen-containing
compounds.
BRAD
BIURET LOWRY
FORD

easier to perform, highest sensitivity higher sensitivity


faster time 1-20 g than Biuret

less susceptible to less susceptible to


chemical chemical simple operation
interferences interferences

more accurate
less chemical linear at wide range
colorimetric
interference of concentrations
analysis

linear at wide range


of concentrations
BRAD
BIURET LOWRY
FORD

more susceptible to
least sensitivity dye is highly
chemical
> 0.5 mg absorbing
interferences

inhibited by very low colorimetric


detergents accuracy

linear at short range


longer time
of concentrations
CONCLUSION &
RECOMMENDATIONS
There are many ways to measure protein
concentration. Three particular
spectrophotometric protein assays were done,
observed, and analyzed in the experiment. The
Biuret assay depends on the interaction of the
peptide bonds to the Cu2+ forming a blue to
violet complex. Lowry assay is the
combination of Biuret assay and the
subsequent reduction with Folin-Ciocalteu
reagent to molybdenum/tungsten blue
complex. Lastly, the Bradford assay is based
on the absorbance shift of Coomassie Blue G-
250 dye as it binds with the protein.
It is recommended to analyze other
spectrophotometric protein assays such as
dye binding and formol titration techniques to
have a comparison on the accuracy,
sensitivity, and convenience in the
quantitative analysis of protein samples.
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