The titration curve is a curve presenting the mobility of each component of the sample against pH, for which the gel containing the carrier amphoteric electrolyte is first isoelectrically focused. Then the gel is turned 90°, the sample is spiked, and the sample is routinely electrophoresed in the direction perpendicular to the pH gradient. This experiment is from "Experimental Techniques in Protein Electrophoresis", edited by Yaojun Guo.

Operation method

titration curve

Materials and Instruments

isoelectric focusing gel

Move

Prepare an isoelectric focusing gel with T=5% and C=3% as described in "Carrier Amphoteric Electrolyte pH Gradient Isoelectric Focusing" and perform isoelectric focusing. A wide pH range (pH 3.5~9.5 or pH 3.5~10 ) should generally be selected in order to have a strong charge on the sample molecules during the second direction of electrophoresis so that the second direction of electrophoresis can be accomplished in a short period of time, to ensure the accuracy of the titration curve as well as to have a complete titration curve for the sample.

At the end of the first direction of electrophoresis, cut off the electrode strip (salt in the electrode strip will interfere with the second direction of electrophoresis ) and turn this gel 90°. Routinely use the electrode strip for polyacrylamide gel electrophoresis in a direction parallel to the sample addition hole, (e.g., for semi-dry electrophoresis), add the sample, and then proceed with the second direction electrophoresis as quickly as possible to avoid diffusion of the pH gradient. In the second direction electrophoresis can be relatively high voltage, electrophoresis time will be greatly reduced.

Since the high salt in the sample and the uneven conductivity of the buffer ions in the second direction electrophoresis can interfere with the results, dialysis or other methods of desalting should be used.

The amount of sample added depends on the size and thickness of the gel, the size of the sample wells and the staining method. If staining with Thomas Brilliant Blue, 5 times the conventional amount is used.

After second direction electrophoresis, the pH gradient is determined with a surface electrode.

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