Functional Characterization of Reductive Dehalogenases by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) (CAT#: STEM-ET-0343-ZJF)

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a very common method implicated for characterization of proteins in their enzymatically active state with a high-resolution separation. In this method, a dye, Coomassie Blue-G250, is used for visualization and induction of external negative charge on the protein complexes. The complexes are then separated based on their molecular weights contrary to that of conventional SDS-PAGE in which the proteins are fractionated based on their charge/mass ratio. In BN-PAGE, the protein complexes tend to migrate according to the pore size of the gradient gel till they reach the pore size limit point. BN-PAGE has a very high resolution ranging from 100 kDa to 10 MDa and it is largely dependent on the concentration range of acrylamide and quality of the gradient gel. BN-PAGE is a simple method holding a great power of result and analysis. It provides a considerable amount of information when coupled with other methods such as LCMS or iTRAQ. It also provides details about the size, subunit composition of the protein complexes, stoichiometry, number and approximate abundance of the complexes/proteins in the sample. If compared with SDS-PAGE in the same gel in the second dimension, BN-PAGE can give precious revelation about the interacting partners of a specific protein as well as its probable subunit composition.

Applied and environmental microbiology, enzyme

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.

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