When a protein is electrophoresed under conditions that keep its natural conformation constant, its mobility can be used as a measure of its homogeneity. Since mobility is a function of both charge and shape, it can be expected to be able to separate the monomeric, dimeric, and other forms from each other. It is also possible to separate conformational isomers of proteins by non-denaturing gel electrophoresis due to differences in isomer shape. This experiment is from Protein Purification and Characterization Lab Guide by Houzhu Zhu.

Operation method

Evaluation of homogeneity experiments by nondenaturing gel electrophoresis mobility shift rate method

Materials and Instruments

Core RNA polymerase Core RNA polymerase-σ32 complex σ32 nondenaturing gel Sample buffer Storage buffer Coomassie Brilliant Blue (CBB) staining solution Decolorizing solution
Non-Denaturing Gel Electrophoresis Unit

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Materials and equipment

Core RNA Polymerase

Core RNA ^{polymerase-σ32} complex, eluted from an immunoaffinity column

^σ32, eluted from a POROS 50S cation exchange column

Non-denaturing gel electrophoresis setup

Reagents

Non-Denaturing Gel Sample Buffer (2X) (Same as SDS Sample Buffer, but without SDS.)

Storage Buffer

Caulmers Brilliant Blue (CBB) Staining Solution

Decolorizing solution

(For the recipe, see "Preparation of Reagents", PP.184~189)

Procedure

1) Prepare non-denaturing gel (e.g., 4%?12% gel) with 10 lanes.
Note: The gradient Tris-glycine minigel for SDS-PAGE sold by several manufacturers (e.g., NOVEX) is actually SDS-free. Frequently, SDS enters the gel from the electrode solution and sample buffer during electrophoresis, and as long as these buffers are free of SDS, these gels can be electrophoresed fairly easily and in a nondenaturing manner.

2) Take 1~10ul of each protein sample, add 5~9ul of storage buffer, then add 10~20ul of 2X non-denaturing gel sample buffer. Mix well. It is recommended that each sample and its appropriate amount of protein be listed in the table below.



3) Add 20ul of sample to each well, run electrophoresis, and decolorize after CBB staining.
Note: Gel exclusion chromatography by high performance liquid chromatography (HPLC) can also be used to determine protein size heterogeneity. As described in the Supplementary Experiments section of the protocol for this module.

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