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Separation of Polypeptides and Analysis of Membrane Protein Complexes by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) (CAT#: STEM-ET-0336-ZJF)

Introduction

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. This service provides an SDS polyacrylamide gel electrophoresis (SDS-PAGE) method for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa.




Principle

Polyacrylamide gel electrophoresis (PAGE) is an electrophoresis which based on polyacrylamide gel. Polyacrylamide gel is tougher than agarose gel and is relatively inert, thermostable, strong, and transparent, with a uniform pore size. PAGE is a widely used technique for separating and analyzing proteins based on their size and charge. It involves the migration of proteins through a porous gel matrix made of cross-linked polyacrylamide under the influence of an electrical field. Known as one of the most versatile and widely used techniques, PAGE has the ability to separate proteins at high resolution, allowing the detection of minor differences in protein composition.
In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), sodium dodecyl sulfate (SDS) is added to PAGE. SDS-PAGE eliminates the influence of structure and charge in electrophoresis, and proteins are separated based on the differences in their molecular weight.

Applications

Molecular biology, polypeptides, membrane protein complexes

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 SDS-polyacrylamide gel