Bio-Rad PROTEAN II XL Cell - Manual (Page 30)

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Note: Buffer leakage during isoelectric focusing can result in damage to the cooling core. It is important
to check for buffer leaks by monitoring both the current and the upper buffer level. During the course of a
normal IEF run, the current decreases as the resistance of the gel increases, the pH gradient is established,
and the upper buffer level is maintained. If a buffer leak should occur, the current will increase, and the level
of upper buffer may decrease. Do not exceed 1,000 V as the maximum focusing voltage.
5. Insert the tube gels into the tube gel adaptor, using the gel tube locator at the bottom of the unit to align
the tubes. Plug any unused tube holes with a stopper.
6. Prepare the sample just before loading. The amount of denaturing sample solution A and/or iso-urea
solution E will depend upon the protein concentration of sample and upon the type of sample. An initial
starting ratio of 1 µl IEF sample concentrate for every 10 µl sample can be used. For denaturing, sam-
ples are heated at 95 °C for 5 minutes then cooled for 2 minutes at room temperature before loading or
before adding iso-urea solution.
7. Load the samples with a Hamilton syringe, or with a Drummond pipet tip. (Generally, 15 to 30 µl of final
diluted sample is loaded.)
8. Carefully overlay the sample with upper electrolyte (20 mM NaOH).