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Analysis of High-Molecular-Weight Proteins by Vertical Agarose Gel Electrophoresis (CAT#: STEM-ET-0320-ZJF)

Introduction

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. This service provides an agarose gel electrophoresis method that has greatly enhanced the resolving power for very large proteins. Agarose electrophoresis is the method of choice for separation and blotting of proteins with very large subunit sizes.




Principle

Gel Electrophoresis is one of the zone electrophoresis, and the supporting medium of it is gel. The gel is a cross-linked polymer, acting as a molecular sieve. Its composition and porosity are chosen based on specific requirements. The types of gel typically used are starch, agarose, and polyacrylamide gels.
Agarose is a purified uncharged polysaccharide extracted from seaweeds. By manipulating the concentration of agarose in the gel, the pore size can be adjusted. Agarose gel electrophoresis is suitable for the separation of proteins larger than 200 kDa, and DNA fragments of 0.2-20kb.

Applications

Molecular biology, proteins

Procedure

1. Preparation: Prepare electrophoresis buffer with a certain pH. Prepare a gel solution of appropriate concentration, taking care not to produce bubbles, and use a cast to solidify the solution into a gel.
2. Sample Application: Put the prepared gel with a cast into the electrophoresis tank, and add an appropriate amount of buffer. Pipette the sample into the sample wells.
3. Electrophoresis: Adjust the appropriate distance between the electrodes. Switch on the electrophoresis apparatus and set the voltage and current. Perform electrophoresis for a period of time.
4. Determination: After electrophoresis, stain the gel for visible results. Observe, record and analyze the position of separated bands. Or, utilize an imaging system for analysis.
5. Downstream processing: After separation, an additional method is often applied to the separated bands for further processing. For example, cut the band out of the gel as a slice to dissolve and purify it.

Materials

• Gel electrophoresis apparatus
• Sample solution
• Buffer solution and agarose gel