| Assay Method Information | |
| | LATS1 Biochemical HTRF Assay |
| Description: | In a 384-well white small volume plate (Greiner 784075) add 3.5 μL of 1× Enzymatic buffer with [5 mM] MgCl2 and [1 mM] DTT. 1× Enzymatic buffer diluted from 5× Enzymatic buffer*. Add 0.5 μL of 20× compound in 100% DMSO. Add 2 μL±[2.8 nM] LATS1 kinase (Carnabio #01-123) in 5× Enzyme Resuspension Buffer (ERB) with [5 mM] MgCl2, [1 mM] DTIT and [5 mg/mL] BSA. 5×ERR prepared from 5× Enzymatic buffer with [25 mg/mL] BSA. Mix plate on plate shaker set at 1,350 rpm for 30 seconds, then centrifuge plate at 1,000 rpm for 1 minute. Incubate for 30 minutes at 25° C. on plate shaker set to 500 rpm. Add 2 μL [12.5 μM] STK Substrate 1-biotin* then add 2 μL [10 mM] ATP. Mix plate on plate shaker set at 1,350 rpm for 30 seconds, then centrifuge plate at 1,000 rpm for 1 minute. Incubate for 40 minutes at 25° C. on plate shaker set to 500 rpm. Stop reaction by adding 10 μL of a 1:1 mix of [625 nM] Streptavidin-XL665 with 1×STK Antibody-Cryptate*. Mix plate on plate shaker set at 1,350 rpm for 30 seconds, then centrifuge plate at 1,000 rpm for 1 minute. Incubate for 60 minutes at 25° C. on plate shaker set to 500 rpm, covered from light. Read plate: ex 330 nm; em1 620 nm and em2 665 nm. Calculate the ratio of the acceptor (665 nm) and donor (620 nm) emission signals for each well. Ratio is equal to signal 665 nm/signal 620 nm×10,000. |
| Affinity data for this assay | |
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