Question

What are the step to do electrophorese 2D in order to purify the protein after cloning ?

Answer 1

2D gel electrophoresis consists of isoelectric focussing with SDS polyacrylamide gel electrophoresis. This separates proteins based on isoelectric point. The isoelectric point is the pH at which protein has no net charge. This is done with an immobilized pH gradient gel strip or with a tube gel containing a low concentration of polyacrylamide. Ampholytes are added to create pH gradient in an electric field and the proteins are loaded. The IEF gel is placed in an electrophoresis system for up to 24 hours and proteins form tight bands at their isoelectric point. The IEF gel is now ready for 2D gel electrophoresis.

The second dimension separates the proteins based on size. There are two parts, the stacking gel which concenterates the sample and the separating gel that is used to separate the proteins. The isoelectric focusing gel is soaked in a solution containing chemical to denature the proteins including sodium dodecyl sulfate - a detergent which gives the proteins a net negative charge. This means that all protein will move in one direction. The IEF gel is then put in the one long well in the stacking gel, sealed in place with agarose and proteins subjected to an electric field to separate. The larger proteins are found at the top and the smaller are found at the bottom of gel. In 2D gel, the proteins appears as spots on gel. These spots can then be further processed or used for mass spectrometry directly.