Measurement Concentration Range; Bradford Kits, Protocols, And Sample Preparation - Thermo Scientific NanoDrop 1000 V3.7 User Manual

Measurement Concentration Range

Unknown protein concentrations from ~100ug/ml up to several
thousand micrograms/ml (ug/ml) can be determined on the NanoDrop
1000 Spectrophotometer using the regular Bradford assay. 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.
Particulates often form with increasing development time, which can
cause significant fluctuations in Absorbance readings. It is also
important to note the total analyte (protein-dye) signal at 595 nm 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
Regular
Bradford
Mini Bradford

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. Use the same pipettor for both volumes to eliminate
any pipette-to-pipette accuracy differences.
Approx. Approx.
Lower Upper
Limit Limit
100 8000
ug/ml ug/ml
15 ug/ml 100 ug/ml
Section 12- Protein Bradford
Typical Reproducibility
(minimum 5 replicates)
(SD= ug/ml; CV= %)
sample range 100-500 ug/ml: ±
sample range 500-8000 ug/ml: ±
sample range 15-50 ug/ml: ± 4
sample range 50-125 ug/ml: ± 5%
12-2
25 ug/ml
5%
ug/ml