User Manual

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4.4 Sample Preparation
In the absence of SDS, the net charge of a polypeptide is determined by its amino acid composition and
the pH of the sample buffer. Only polypeptides with a net negative charge migrate into Criterion gels
under native conditions. Most polypeptides have an acidic or slightly basic pI (~3–8). These proteins can
be separated using the following standard protocol:
1. Determine the protein concentration and load volume of your sample based on the detection
method used (see Chapter 12 for approximate stain sensitivities).
2. Dilute the sample in twice the volume of native sample buffer (DO NOT HEAT SAMPLES).
For example, combine: 5 μl sample
10 μl native sample buffer (catalog #161-0738)
15 μl total volume
Strongly basic proteins (pl >8.5) have a net positive charge and will not enter a Criterion gel under native
conditions. To allow polypeptides with a net positive charge to migrate into a native Criterion gel, change
the polarity of the electrodes by reversing the color-coded jacks when connecting to the power supply.
4.5 Running Conditions
Running conditions for native PAGE are similar to the standard running conditions used for SDS-PAGE
(see Section 3.5). If high temperature is a concern, run native PAGE at lower voltage; at lower voltages, runs
require more time to complete.
Table 4.1. Running conditions for native PAGE for Criterion gels in the Criterion cell.
Laemmli/Laemmli-like Tris-Acetate
Running buffer Native Native
Power conditions 200 V constant 200 V constant
Expected current (per gel)
Initial 90–120 mA 70–80 mA
Final 35–55 mA 25–35 mA
Run time 55 min 75 min
Criterion Precast Gels