Section 12- Protein Bradford
12. Protein Bradford
The Bradford Assay is a second alternative method commonly utilized for determining protein concentration. It is often
used for more dilute protein solutions where lower detection sensitivity is needed and/or in the presence of components
that also have significant UV (280 nm) absorbance. Like the BCA and Lowry methods, the Bradford assay requires a
standard curve to be generated before unknown proteins can be measured.
The Bradford uses the protein-induced absorbance shift of Coomassie Blue dye to 595 nm as a measure of protein
concentration. The bound protein-dye complex is measured at 595 nm and normalized at 750 nm. A single stabilized
reagent mixture containing Coomassie Blue dye, alcohol, and surfactant in kit form is available from numerous
manufacturers. Follow the respective manufacturer’s recommendations for all standards and samples (unknowns),
ensuring they are subjected to the identical conditions and timing throughout the assay.
Sample Volume Requirement
Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be
measured. Additionally the presence of surfactants or detergents in reagents, such as the Bradford reagent, can
significantly alter surface tension. This occurrence can be overcome without affecting the sample’s absorbance by using a
larger sample volume.
A 2 ul sample size is recommended for protein measurements
.
Pedestal Reconditioning
Proteins and solutions containing surfactants are known to “un-condition” the measurement pedestal surfaces so that the
liquid column does not form. If this occurs, “buff” the measurement pedestal surfaces by rubbing each measurement
surface aggressively with a dry laboratory wipe 30-40 times. This will “re-condition” the surface allowing the liquid sample
column to form. Alternatively, use the NanoDrop Pedestal Reconditioning Compound (PR-1) as a rapid means of
reconditioning the pedestals when the surface properties have been compromised and liquid columns break during
measurement. Additional information about the PR-1 kit may be found at
www.nanodrop.com.
Measurement Concentration Range
On the NanoDrop
®
ND-1000 Spectrophotometer using the regular Bradford assay, unknown protein concentrations from
~100ug/ml up to several thousand micrograms/ml (ug/ml) can be determined. The best linearity is in the 100-1000 ug/ml
range. A mini-Bradford assay covers the approximate range of 15-125 ug/ml.
Coomassie dye-dye and Coomassie dye-protein aggregates are frequently encountered in Coomassie dye-based protein
assays. With time, particulate can be observed, which can cause significant fluctuations in Absorbance readings. It is also
important to note the total analyte (protein-dye) signal at 595nm is limited to ~ 0.150 A as a result of the 1.0mm pathlength
of the instrument, the Bradford (Coomassie dye) reagent concentration, and the acidic pH. Making measurements in
triplicate
of standards and samples (unknowns) is good practice, particularly with the limited assay signal obtained with
the Bradford Assay.
Assay
Type
Approx.
Lower
Limit
Approx.
Upper
Limit
Typical Reproducibility
(minimum 5 replicates)
(SD= ug/ml; CV= %)
Regular Bradford
100 ug/ml
8000 ug/ml
sample range 100-500 ug/ml:
±
25 ug/ml
sample range 500-8000 ug/ml:
±
5%
Mini Bradford
15 ug/ml
100 ug/ml
sample range 15-50 ug/ml:
±
4 ug/ml
sample range 50-125 ug/ml:
±
5%
Bradford Kits, Protocols, and Sample Preparation
Commercial Bradford Protein kit manufacturers typically outline procedures for two different concentration ranges:
•
A regular assay – using a 50:1 reagent / sample volume ratio. To accurately prepare standards, we suggest using a
minimum sample volume of 4 ul in 200 ul of Bradford reagent (larger sample volume is preferable).
•
A mini assay– using a 1:1 reagent / sample volume ratio. To prepare sufficient volume of these 1:1 mixtures, we
suggest using a minimum of 10 ul of sample and 10 ul of Bradford reagent in a PCR tube. Using the same pipettor for
both volumes will eliminate any pipette-to-pipette accuracy differences.
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