Protein binding assay with immunoaffinity mediators

Summary

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

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.

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