Section 10- Protein BCA
10. Protein BCA
The BCA (Bicinchoninic Acid) Protein Assay is an alternative method for determining protein concentration. It is often used
for more dilute protein solutions and/or in the presence of components that also have significant UV (280 nm) absorbance.
Unlike the Protein A280 method, the BCA Assay requires a standard curve to be generated each time it is run, before
unknown proteins can be measured. The resulting Cu-BCA chelate formed in the presence of protein is measured at its
wavelength maximum of 562 nm and normalized at 750 nm. Pre-formulated reagents of BCA and CuSO
4
, utilized in the
assay, are available in kit form from numerous manufacturers. Follow their recommendations when mixing the respective
reagents at the time the assay is to be performed.
Sample Volume Requirements
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 a
t
www.nanodrop.com.
Measurement Concentration Range
On the NanoDrop
®
ND-1000 Spectrophotometer, the regular BCA assay can measure samples from ~0.20 mg/ml up to
8.0 mg/ml. A mini-BCA assay covers an approximate range of 0.01 – 0.20 mg/ml.
Assay
Type
Approx.
Lower
Limit
Approx.
Upper
Limit
Typical Reproducibility
(minimum 5 replicates)
(SD= mg/ml; CV= %)
Regular BCA
0.2 mg/ml
8.0 mg/ml
±
2% (over entire range)
Mini BCA
0.01 mg/ml
0.20 mg/ml
±
0.01 mg/ml (over entire range)
BCA Kits, Protocols, and Sample Preparation
Commercial BCA Protein kit manufacturers typically outline procedures for two different protein concentration ranges:
•
A regular assay – using a 20:1 reagent / sample volume ratio. To accurately prepare standards, we suggest using a
minimum sample volume of 4 ul in 80 ul of BCA 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 BCA reagent in a PCR tube. Using the same pipettor for
both volumes will eliminate any pipette-to-pipette accuracy differences.
Note: If you run the assay at 60
°
C, doubling the volumes may afford greater insurance against skewed results from
evaporation / condensation within the sealed reaction tube.
In addition to the kit reagents, protein standards (BSA) for generating a standard curve are provided for the BCA method
by the manufacturer. Follow the manufacturer’s protocol for the assay including recommended incubation times and
temperature. Additionally, use the respective standard (e.g., BSA) and dilutions that cover the analytical range (mg/ml) of
interest. Note: Since the ND-1000 can measure higher protein concentrations, you may need to supply your own protein
standards at higher concentrations than provided by the manufacturer.
Unique Screen Features
View Standard Curve (F8):
selecting this button allows the user to view the standard curve at any time.
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