Modulus™
Single Tube Multimode Reader Method for
Coomassie Plus - The Better Bradford™ Assay Kit
1. INTRODUCTION
The Modulus™
by Turner BioSystems in combination with the Pierce's Coomassie Plus™
Assay kit provides a convenient procedure for quantifying protein. When
the Coomassie dye binds protein the absorption maximum shifts from 465
nm to 595 nm. The Absorbance Module detects as little as 25 µg of
BSA in 1.5 mL of Coomassie Plus Reagent.
Figure
1. Bradford Assay was performed on the Modulus using the Absorbance
Module, the 600 nm filter, and the Coomassie Plus Reagent. Each point
represents the average of replicate samples after background subtraction
(n=2).
2. MATERIALS
·
Modulus (P/N 9200-000 or 9200-002)
· Absorbance Module (P/N 9200-050)
· 600 nm filter paddle (P/N 9200-052)
· 10 x 10 mm disposable methacrylate cuvettes (7000-959)
· Coomassie Plus - The Better Bradford Assay kit (Pierce catalog
#'s 23225 and 23227)
3. PREPARATION
NOTE: Store assay reagent at 4°C. Unopened vials of bovine serum albumin
(BSA) standard may be stored at room temperature.
3.1 BSA
Standard Curve
Prepare a serial dilution of BSA that covers the range for your samples.
For example, create a two-fold dilution series from 1000 µg/mL to
125 µg/mL. Make sure to include a blank solution (diluent only)
in your standard curve preparation.
NOTE: Prepare the dilution series in the same diluent as the samples
for best results.
3.2 Coomassie Plus Reagent
Determine the total volume of reagent required. Each sample and standard
requires 1.5 mL of reagent.
3.2.1 Gently invert the bottle of Coomassie Plus Reagent solution to mix the
solution before removing the amount necessary for the assay.
3.2.2 Equilibrate the reagent to room temperature before use.
NOTE: Coomassie Plus Reagent may form dye-dye and dye-protein aggregates
when left undisturbed. Gentle mixing dissolves the aggregates. For best
results, mix the reagent before dispensing and again before measuring
absorbance.
3.3 Samples
3.3.1 For each standard or sample, pipette 50 µL into an
individual 10 x 10 mm methacrylate cuvette.
3.3.2 Add 1.5 mL of the Coomassie Plus reagent to each cuvette.
3.3.3 Incubate the samples and standards for 10 minutes at room
temperature.
3.4 Instrument
Setup
3.4.1 Power OFF the Modulus. Install the Absorbance Module into the sample compartment
according to Operating Manual.
3.4.2 Insert the 600 nm filter paddle in the Absorbance Module.
3.4.3 Turn ON the Modulus and use the touchscreen to choose the
Absorbance operation mode.
3.4.4 Touch "Calibrate" and use the black cuvette to set the Modulus
to calibrate the zero (dark) reading.
3.4.5 Use a cuvette containing 2 mL of ultrapure water to calibrate the baseline
(100% transmittance) reading.
3.4.6 Touch "OK" to accept the calibrations and return to the
"Home" screen.
4. SAMPLE
ANALYSIS
4.1 Insert the sample or standard into the Absorbance Module and
touch "Measure Absorbance" to commence measurement.
4.2 Record the results in Absorbance units (Ab).
4.3 Use a standard curve to determine the protein concentration of
each unknown sample. A four-parameter (quadratic) or best-fit curve provides
the best accuracy.
5. ABOUT PIERCE
Coomassie-Plus - The Better Bradford is a registered trademark of Pierce
Biotechnology, Inc.
Orders for Pierce products may be placed by:
Phone: (800) 874-3723
Fax: (800) 842-5007
Internet: www.piercenet.com
Mailing Address:
3747 N. Meridian Road
P.O. Box 117
Rockford, IL 61105
USA
6. ABOUT
TURNER BIOSYSTEMS
Modulus is
a trademark of Turner BioSystems. Orders for Turner BioSystems' products
may be placed by:
Phone: (408)
636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919
Contact us
via our contact
form
Internet: www.turnerbiosystems.com
Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085
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