Blue Native PAGE Protocol
The experimental procedure for Blue Native PAGE (BN-PAGE) mainly involves the following steps:
1. Sample Preparation
Cells are lysed using mild detergents, such as digitonin or n-dodecyl β-D-maltoside, at 4°C to isolate protein complexes for subsequent analysis.
2. Protein Extraction
Cellular debris and remnants are removed by centrifugation, and the resulting supernatant, containing the protein extract, is collected.
3. Sample Buffering
The protein sample is mixed with an appropriate amount of G-250 dye to prevent the dissociation of native protein complexes and to enhance their negative charge for electrophoresis.
4. Electrophoresis
Electrophoresis is conducted on a 4-16% polyacrylamide gradient gel under non-denaturing conditions, with a cooling system maintained at low temperatures to preserve the integrity of the protein complexes.
5. Staining and Analysis
After electrophoresis, the protein bands are visualized by staining with Coomassie Brilliant Blue. The bands can then be further analyzed using mass spectrometry to identify the proteins.
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