14 Objectives for Week 4
- Determine the concentration of the pure, desalted wild-type protein samples.
- Determine if wild-type protein is active.
- Run SDS-PAGE of crude extracts of ncAA-mutant proteins expressed in Week 3.
- Run SDS-PAGE of purified wild-type proteins.
- Purify ncAA-mutant proteins.
- Develop kinetic assay and determine catalytic constants for wild-type and ncAA-mutant enzymes (see below for advice on running assays).
Determining Concentration of Protein
- Coomassie (Bradford) Protein Assay Kit, containing
- Coomassie Protein Assay Reagent
- Bovine Albumin Standard aliquots (2 mg/mL)
Determine activity and catalytic constants of Carbonic Anhydrase and Catalase
Materials will vary by group depending upon the nature of the assay. Each group is responsible for ensuring that the necessary equipment and materials are available from week to week for executing their assays.
Suggested Resources and Protocols
- Protein Concentration: The manual for the Bio-Rad Bradford Assay can be found online. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
- SDS-PAGE: Use resources from Week 3.
- Enzyme Assay: See Weeks 5-9 for details about assay resources.