Bio-Rad Ready Gel Precast Gels for 2-D Electrophoresis - Manual (Page 17)

4.5 Sample Preparation
Determine the desired protein concentration and load volume of your sample based on the detection
method used. (See section 10.1 for approximate stain sensitivities). 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 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 (see section
4.4; DO NOT HEAT SAMPLES).
Strongly basic peptides (pl >9) will have a net positive charge in a native PAGE Tris-HCl gel. In order for
polypeptides with a net positive charge to migrate into a native PAGE Tris-HCl gel, the polarity of the
electrodes must be changed by reversing the color-coded jacks when connecting to the power supply.
4.6 Running Conditions
Power conditions 200 V constant
Starting current:
50 mA/gel
Final current: 30 mA/gel
Run time 35 min
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