Bio-Rad Gel Doc™ EZ System - Manual (Page 35)

Native PAGE
21
Native PAGE Buffers
Sample Preparation
Determine the desired protein concentration and load volume of your sample
based on the detection method used. Sample preparation for Native PAGE
applications requires special consideration. In the absence of SDS, the net
charge of a polypeptide will be determined by the pH of the sample buffer.
Only polypeptides with a net negative charge will migrate into a native PAGE
Criterion Tris-HCl gel. Most polypeptides have an acidic or slightly basic pl
(~3–8). These proteins can be separated using a standard protocol by diluting
1 part sample with 1 part native sample buffer (DO NOT HEAT SAMPLES).
Strongly basic peptides (pl >9) will have a net positive charge in a Native
PAGE Criterion Tris-HCl gel. In order for polypeptides with a net positive
charge to migrate into a Native PAGE Criterion Tris-HCl gel, the polarity of the
electrodes must be changed by reversing the color-coded jacks when
connecting to the power supply.
Running Conditions
Running buffer, 1x 25 mM Tris
192 mM glycine
DO NOT ADJUST pH
Sample buffer 62.5 mM Tris-HCI, pH 6.8
25% glycerol
0.01% Bromophenol Blue
Power conditions 200 V constant
Starting current- 90-120 mA/gel
Final current- 35-55 mA/gel
Run time 55 minutes