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Protein Assays
Principles behind dye-based
absorbance and colored complex
measurements for protein
quantification.
OBJECTIVES
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.
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.
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
more accurate
less chemical linear at wide range
colorimetric
interference of concentrations
analysis
more susceptible to
least sensitivity dye is highly
chemical
> 0.5 mg absorbing
interferences