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Technical Manual
Contents
General Information ............................................................................................ 1
Contents of the Kit .............................................................................................. 1
Storage .............................................................................................................. 1
How to Use Protein Quantification Kit - Rapid .................................................... 1
Required equipments and materials ......................................................... 1
Protocol .................................................................................................... 1
Materials that interfere with the assay ...................................................... 2
Protein-to-protein variation ....................................................................... 3
Notes ................................................................................................................. 3
Product Code and Price ......................................................................................3
Related Product ................................................................................................. 3
References ......................................................................................................... 3
General Protocol at a Glance - standard assay .................................................. 4
General Protocol at a Glance - high sensitivity assay ......................................... 5
PROTEIN QUANTIFICATION KIT-Rapid
Product Code: PQ01-10 (500 tests)
PQ01-12 (2500 tests)
Revised April 26, 2006
For the most up-to-date technical manual, access www.dojindo.com/tm - 1 -
for 12 months at 0-5
o
C and 6 months at room
temperature. Though the plastic surface of the CBB
Solution bottle may gradually be stained, the
background OD of CBB Solution does not increase
during the storage at 0-5
o
C.
HOW TO USE THE KIT
STORAGE
1. Required Equipments and Materials
Microplate reader (600 nm filter)
96 well microplates
10
µ
l, 100-200
µ
l pipettes, multi channel pipette
1.5 ml tubes
2. Protocol
Standard Assay (for microplate reader)
1) Dilute BSA Standard Stock Solution (4000
µ
g/ml)
with ddH
2
O to prepare various concentrations of
BSA Standard solution by multiple dilutions.
2000
µ
g/ml, 1000
µ
g/ml, 500
µ
g/ml, 250
µ
g/ml,
125
µ
g/ml, 63
µ
g/ml, 32
µ
g/ml, 0
µ
g/ml
2) Add 6
µ
l of various concentrations of BSA Standard
solution or sample solution to each well. The use
of 3 wells per one standard BSA solution or sample
solution (n=3) is recommended.
3) Add 300
µ
l CBB Solution to each well.
Be careful not to introduce bubbles to the wells
since the bubbles interfere with the O.D. reading.
4) Leave the plate for 1 min. at room temperature.
5) Immediately, measure the absorbance of each well
at 570-600 nm using a microplate reader.
6) Subtract the absorbance of the blank solution from
the absorbance of each well.
7) Plot the concentration of BSA on the X-axis and
value of the absorbance on the Y-axis to prepare
a calibration curve (Fig. 3). Determine protein
concentrations of unknown samples using the
calibration curve.
High Sensitivity Assay (for microplate reader)
This assay is used for purified protein detection.
a)
1) Dilute 100
µ
g/ml BSA Standard Solution with ddH
2
O
to prepare various concentrations of BSA Standard
solution by multiple dilutions (stock concentration
of BSA Standard Solution is 4000
µ
g/ml).
50
µ
g/ml, 25
µ
g/ml, 12.5
µ
g/ml, 6.3
µ
g/ml,
3.2
µ
g/ml, 1.6
µ
g/ml, 0.8
µ
g/ml, 0
µ
g/ml
2) Add 150
µ
l of various concentrations of BSA
Standard solution or sample solution to each well.
The use of 3 wells per one standard BSA solution
or sample solution (n=3) is recommended.
3) Add 150
µ
l CBB Solution to each well, and mix.
Be careful not to introduce bubbles to the wells
since the bubbles interfere with the O.D. reading.
4) Leave the plate for 1 min. at room temperature.
5) Immediately, measure the absorbance of each well
at 570-600 nm using a microplate reader.
GENERAL INFORMATION
For protein concentration determination, several
methods are available, such as Lowry method,
bicinchoninate method (BCA method), Biuret method
and Bradford method. Coomassie Brilliant Blue G (Fig.
1) has been utilized for quick and sensitive protein
detection known as the Bradford method. Coomassie
Brilliant Blue G interacts with protein and stains blue
under acidic conditions. The maximum change in the
absorbance by interaction with protein is at 595 nm
(Fig. 2). The staining reaction completes within 1 min.
and the color is stable for 30 min. Therefore, protein
concentration can easily be determined within a few
minutes by colorimetric detection. This kit contains
ready-to-use Coomassie Brilliant Blue G solution and
BSA solution as a protein standard solution and is
suitable for microplate assay. The protein detection
range is from 10
µ
g/ml to 2000
µ
g/ml by standard
method, and is from 0.1
µ
g/ml to 50
µ
g/ml by micro
method. Since the sensitivity of CBB-based protein
assay depends on the type of proteins, please note
the protein-to-protein variation in quantification.
Figure 1. Structure of Coomassie Brilliant Blue G
Figure 2. Absorption spectrum of CBB with and without protein
a) protein free
b) 500
µ
g BSA/ml
CONTENTS OF THE KIT
PQ01-10 (500 tests):
CBB Solution ................................... 100 ml x 2 bottles
BSA Standard Solution (4000
µ
g/ml) ... 1.5 ml x 1 vial
PQ01-12 (2500 tests):
CBB Solution .................................. 1000 ml x 1 bottle
BSA Standard Solution (4000
µ
g/ml) ..1.5 ml x 2 vials
STORAGE
Store the kit at 0-5
o
C. CBB Solution is stable
N
C
2
H
5
N
C
2
H
5
HN
SO
3-
-
O
3
S
+
Na
+
CH
3
CH
3
OC
2
H
5
500
600
700
800
0
0.4
0.8
0.2
0.6
Wavelength/ nm
a)
b) - 2 -
8) Transfer the mixed solution to a cell, and measure
the absorbance of the solution at 600 nm using a
spectrophotometer.
9) Determine the protein concentration of the sample
solution using the calibration curve.
3. Materials That Interfere with the Assay
Maximum non-interfering concentrations of
materials, which may interfere with the protein
quantification using Protein Quantification Kit - Rapid
(standard method), is shown in Table 1. Since the assay
system is based on the interaction of hydrophobic sites
of protein, detergents give positive error.
Table 1 Compatible concentrations
a)
of possible interfering
materials with CBB assay
Chemical
Sample Concentration
Detergent
Brij 35 .............................. 0.125%
Brij 56 ............................. 0.025%
Brij 58 ............................. 0.005%
Triton X-100 ..................... 0.125%
Triton X-114 ..................... 0.125%
Tween 20 ......................... 0.25%
Tween 80 ......................... 0.1%
SDS ................................. 0.1%
CHAPS ............................ 4%
CHAPSO .......................... 4%
MEGA 10 .......................... 4%
Octyl- -D-glucoside ..........0.5%
Organic solvent
Ethanol ............................ 10%
Isopropanol .......................10%
DMSO .............................. 10%
Chelating agent
EDTA ................................ 0.4 M
DTPA ............................... 0.4 M
Salt
Sodium chloride ............... 2 M
Potassium chloride .......... 2 M
Sodium acetate ................ 0.4 M
Sodium bicarbonate ......... 0.1 M
Buffer
Citrate pH 5.0 ................... 0.125 M
MES pH 6.1 ..................... 0.125 M
Tris pH 7.4 ....................... 0.0625 M
PBS .................................. no interference
HEPES pH 7.5 ................. 0.125 M
CHES pH 9.0 ................... 0.125 M
Reducing agent
Glucose ............................ 2 M
Glutatione ........................ 0.04 M
Ascorbic acid ................... 0.4 M
Dithiothreitol (DTT) ......... 0.01 M
2-Mercaptoethanol ......... 1.3 M
BACKGROUND CONTROL
a) The compatible concentration (in sample solution) was
determined within +5% fluctuation of the slope of BSA
calibration curve.
6) Subtract the absorbance of the blank solution from
the absorbance of each well.
7) Plot the concentration of BSA on the X-axis and
value of the absorbance on the Y-axis to prepare
a calibration curve. Determine protein
concentrations of unknown samples using the
calibration curve.
a: Detergents severely interfere with micro assay.
Figure 3. Typical calibration curve prepared using BSA
Standard Solution.

Standard Assay (for spectrophotometer)
1) Prepare BSA Standard solutions as indicate in the
protocol on page 1 for Standard Assay.
2) Add 100
µ
l of a BSA Standard solution to a test
tube.
3) Add 2.5 ml CBB Solution to the same test tube.
4) Transfer the mixed solution to a cell, and measure
the absorbance of the solution at 600 nm using a
spectrophotometer.
5) Repeat the procedure 2 - 4 for the rest of BSA
Standard solutions, and prepare a calibration
curve.
6) Add 50
µ
l of a sample solution to a test tube.
7) Add 2.5 ml CBB Solution to the same test tube.
8) Transfer the mixed solution to a cell, and measure
the absorbance of the solution at 600 nm using a
spectrophotometer.
9) Determine the protein concentration of the sample
solution using the calibration curve.

High Sensitivity Assay (for spectrophotometer)
1) Prepare BSA Standard solutions as indicate in the
microplate reader protocol on page 1 for High
Sensitivity Assay.
2) Add 1.5 ml CBB Solution to a test tube.
3) Add 1.5 ml of a BSA Standard solution to the tube,
and mix.
4) Tr