Whether you are conducting your first topo I specific assay or you have multiple publications identifying novel topoisomerase inhibitors, you are aware that working with catalytically active proteins can involve extra time constraints if you do not first validate the working conditions of your assay. The following simple experiment should be performed when you receive your human topoisomerase I enzyme and topoisomerase I assay kit. You must show that in your lab, you can detect topo activity. This experiment is easy and validates that all components are working in your lab.
ENZYME ACTIVITY TITRATION
1. Topo I Testing: Set up a series of reactions as follows:
2. Add the components in the order shown (enzyme last) and assemble reactions on ice.
3. Incubate for 30-60 minutes at 37°C.
4. Stop the reaction with 5x stop buffer per the manual.
5. Prepare 1% agarose gel. Do not include Ethidium Bromide in your gel. Note that is is very important to avoid Ethidium Bromide – EB will make it difficult to resolve products.
6. Load the gel, avoid using outside lanes which may skew the DNA bands.
7. Run gel at 2.5v/cm (distance between electrodes) until the blue dye front has migrated roughly 60-75% down the gel.
8. Remove gel and stain with 0.5ug/ml ethidium bromide for 15-20 minutes.
9. Destain for 10 minutes with water (change water once or twice with gentle agitation on a rotating platform).
10. Take a picture of the gel. Here is what typical reaction products in a gel cast without EB should look like:
**Nicked DNA will be present in all supercoiled DNA substrate and can be ignored. It tends to co-migrate near relaxed DNA products.
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