Protein binding assay with immunoaffinity mediators
Protein binding assay with immunoaffinity mediators
This experiment describes how much time it takes for a protein to bind to an immunoaffinity medium. This experiment is from the Protein Purification and Identification Lab Guide by Houzhu Zhu.
Operation method
Protein binding assay with immunoaffinity mediators
Materials and Instruments
SDS-PAGE electrophoresis device Polyacrylamide small gel Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SDS-PAGE electrophoresis device
Polyacrylamide mini-gel (10%)
Operating Procedures
1) Mix the dissolved, diluted AMS precipitate and washed immunoaffinity medium and take 100-ul samples at 0, 5, 15, and 30 min each.
2) Immediately centrifuge the sample and leave the supernatant for SDS-PAGE electrophoretic analysis to determine the binding of the core RNA polymerase-σ32 complex to the antibody.
Note: Free σ32 does not bind to the immunoaffinity column, whereas the core RNA polymerase complex in the supernatant does bind and can be detected by the removal of the core β'β subunit (~155 kDa double band on SDS gel) from the supernatant.