| Solution Information | help | |
| Enzyme: | Alkaline phosphatase, tissue-nonspecific isozyme | |
| inhibitor: | BDBM60707 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1. TNAP - provided by Dr. Jose Luis Millan 2. CDP-Star (New England Biolabs # N7001S) 3. TNAP buffer - 200 mM DEA, 0.04 mM ZnCl2, 2 mM MgCl2 Protocol 1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 and 44-48 (Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Compounds are transferred from a 10 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations. 2. Add 2 uL/well of TNAP (1:200 dilution in TNAP buffer) (columns 3 through 48) a. For negative control add 2 uL of TNAP buffer instead of TNAP to columns 1 and 2 3. Add 2 uL/well of CDP-Star (85 uM in MQ water) to all wells 4. Spin the plate down to maintain an even level of volume 5. Cover the plate and incubate the plate at RT for 30 minutes 6 | |
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