Protein Protocols Protein Protocols

260 Choi, Hong, and Yoo
Table 1
Linearity of CBB and NN Staining for Four Purified Proteins
2. Materials
2.1. Equipment
1. Slab-gel apparatus.
2. Power supply (capacity 600 V, 200 mA).
3. Boiling water bath.
4. Gel dryer and vacuum pump.
5. Plastic container.
6. Rocking shaker.
Slope b y-intercept b Correlation coefficient b
Proteina A B A B A B
BSA 14.1 10.1 13.2 6.4 0.986 0.994
OVA 8.7 7.1 2.7 1.4 0.993 0.998
G-3-P DHase 8.0 5.8 5.3 2.2 0.997 0.999
CA 15.4 13.5 12.5 8.8 0.986 0.997
a Proteins were separated on 12.5% polyacrylamide gel, and densities and band area were
determined with computerized densitometer. Some of the data are illustrated in Fig. 1. The
range of amount of proteins was 0.25–12.5 µg. The number of points measured was six (0.25,
0.5, 1.0, 2.5, 5.0, and 12.5 µg).
b Slopes, y-intercepts, and correlation coefficients were determined by linear regression
analysis. A, CBB staining; B, NN staining.
2.2. Solutions
1. Destaining solution (1.0 L): Mix 530 mL distilled water with 400 mL methanol and 70 mL
glacial acetic acid.
2. Simultaneous staining solution (1.0% [w/v] NN): Dissolve 1.0 g NN (pure NN * without
K2SO4) in 100 mL reservoir buffer. Stir until fully dissolved at 50–60°C (stable for months
at 4°C) (see Note 7).
3. Poststaining solution (0.05% [w/v] NN): Dissolve 0.05 g NN (pure NN * ) in 100 mL
destaining solution. Stir until dissolved thoroughly (store at room temperature).
3. Methods
3.1. Simultaneous Staining
The method is based on the procedure of Borejdo and Flynn (14), and is described
for staining proteins in a 7.5% SDS-polyacrylamide gel.
1. Electrophorese the samples for 10 min to allow protein penetration into the upper gel phase.
2. Turn off the power, and then add 1% NN dissolved in reservoir buffer to the upper reservoir
buffer to give a final concentration of 0.01–0.015% NN.
3. Stir the reservoir buffer sufficiently to ensure homogeneity.
4. Resume electrophoresis.
* NN diluted with 100- to 200-fold K2SO 4 has been used as an indicator for the determination of
calcium in the presence of magnesium with EDTA (see Note 7).