TopoGEN has extensive experience with assays for topoisomerase inhibition in vivo. This analysis allows the investigator to ascertain whether a novel agent is active against endogenous Top2bin whole cells. An important benefit to this analysis is that one can use any tumor cell or tissue in order to establish clinical efficacy against a specific tumor cell line. We use essentially the same basic approach for Topo I, and Topo IIa,b. The kit is based upon ‘band shifting’ in a western blot using mono-specific antibody probes for Top2a (TG1024-A) or Top2b (TG1024-B.
This kit can be used to unambiguously detect a Top2a, or Top2b-specific poisoning in the context of a live cell. It is specific for Top2 poisons and not catalytic inhibitors. In addition, the kit can be used to profile the sensitivity of any given cell line to VP16 or other established Topoisomerase poisons.
Cat TG1024-A: A rabbit polyclonal AB is provided with this kit (TG2011-1). This AB is provided at 2.5 units/ul (2ul of AB will make 5mL of working probe). A total of 50ul of stock AB is provided with this kit.
Cat TG1024-B: A monoclonal AB is provided with this kit. This AB is provided at 2.5 units/ul (2ul of AB will make 5mL of working probe). A total of 50ul of stock AB is provided with this kit.
IMPORTANT: Cat 1024A uses a rabbit polyclonal and 1024B uses a mouse monoclonal AB as probe. This will dictate which secondary AB to use.
The following reagents are common to both 1024-A and 1024-B:
-Etoposide (Lyophilized) resuspend in 0.25ml DMSO to give 10 mM Stock(to replenish see Cat TG4140).
-Glycerol (100%) (1ml)
-10x PBS Stock (12ml in 15mL conical tube)
-10x NE1 Stock (3ml in a 5ml microfuge tube). Make 1x fresh for each experiment.
-10x NE2 Stock (1ml in a 1.5 ml microfuge tube). Make 1x fresh for each experiment. ***There is no glycerol in the 10x stock, you must add to the 1x stock to 10% (100% glycerol is provided for this purpose).
Not Included in this kit (and required):
-Equipment and supplies for Western Blotting (gel units, power supply, running buffer, SDS loading buffer plus dye, membranes, transfer units, misc. buffers, dry milk blotto, etc.).
-Cell culture related equipment and supplies.
-Chemiluminescent Detection reagents (HRP and related).
-Secondary AB reagents.
-Dounce homogenizer (2ml) with tight fitting pestle.
Ships on blue ice. Store this kit at 4°C upon receipt.
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