| Assay Method Information | |
| | Inhibitory Activities Against HSET-HSET ADP-Glo Assay |
| Description: | Reagents: (+4° C. Storage)Paclitaxel Prod.No.TXD.01 2 mM in DMSO (from Universal Biologicals Cambridge).Tubulin Protein (Pre-formed Microtubules): Bovine Brain Prod.No.MT001-XL Lot.025 10 mg/mL (from Universal Biologicals Cambridge).Reagents: (−80° C. Storage)MT001-XL—Tubulin Protein (Pre-formed Microtubules): Bovine Brain—reconstituted in buffer—15 mM PIPES pH7, 1 mM MgCl2, 20 μM paclitaxel (aliquots at 10 mg/mL) from Universal Biologicals Cambridge.HSET full length protein—Current batch is FL HSET Prep1 (SEQ-000096_002-01_01)Buffer is 20 mM Hepes pH 7.5, 200 mM NaCl, 2 mM TCEP, 5% glycerol (5 μL aliquots at 10.2 μM)Reagents: (−20° C. Storage)ADP-Glo Kinase Assay kit (Promega Prod.No.V9102) 10,000 assay pointsADP-Glo reagent (50 mL), Kinase Detection Reagent (100 mL), and Ultrapure ATP (10 mM) aliquotedBuffer Stocks (filtered and stored at rt for up to 1 month)HEPES acid, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid MW:238, 238.3 mg/mL=1 M7149 mg/30 mL=1M (pH to 6.8 with 5 M NaOH)PIPES, 1,4-Piperazinediethanesulfonic acid MW: 302.4, 30.24 mg/mL=100 mM; 907.2 mg/30 mL=100 mM (pH to 7 with 5 M NaOH, white cloudy suspension until the pH changes)EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid MW: 380.35, 38.035 mg/mL=100 mM1901.75 mg/50 mL=100 mM (pH to ˜7 with 5 M NaOH)—takes a long time to get into solution and for pH to stabilizeTriton-X-100, MW: 625, 62.5 mg/mL=100 mM (6.25% w/v) (viscous, pipette Triton X-100 with a Gilson using a trimmed pipette tip)ECHO ProtocolCreate an ECHO intermediate plate by adding 24.5 μL DMSO to columns 1 & 2, and 40 μL DMSO to columns 23 & 24 of an ECHO 384PP plateAdd 100 nL of compound/DMSO per well in 384-well Proxi-Plate Plus (white) —Perkin Elmer Cat #6008289 using ECHO dose response protocol 100 nL normal to Assay buffer (Keep on ice): HEPES pH 6.8, MgCl2, EGTA, Triton X-100, DTT, HPLC H2O Microtubule Working Solution (3.2):1 ml PM buffer: PIPES pH 7.0, MgCl2, HPLC H2O, Paclitaxel, mix well, store at rtThen add 26.4 μL of 10 mg/ml microtubules to 724 μL of the above PM buffer to make 750 μL of microtubule working solution @350 μg/mL microtubules (store at rt)Stock HSET enzyme solution (3.1)—keep on iceAdd 1.33 μL 10.2 μM HSET Prepi (SEQ-000096_002-01_01) to 1358 μL assay buffer to make a 10 nM stock for a 5 nM final assay concentration (1/1020 dilution).HSET/Microtubule working solution (3.3)Mix solutions 3.1 and 3.2 in the ratio of 2.5:1 (1214.3 μL 3.1+485.7 μL 3.2) keep at rt for 15 min3.2 is diluted 3.5-fold, 3.1 is diluted 1.4-fold BLANK solution is 357 μL 2XAB+143 μL microtubule working solution 3.2. (Same proportions as HSET/Microtubule working solution (3.3))ATP Working solution is 1 μL 10 mM UltraPure ATP (Promega kit)+999 μL ddH20 gives 10 μM ATP for a 3 μM final assay concentration, stored on ice.Assay procedure in PROXIPLATE 384 PLUS WHITE (Perkin Elmer) plates (Remove 2.4 mL ADP Glo and 4.5 mL Kinase detection reagent from freezer to warm up to rt)Add 3.5 μL BLANK solution to assay plate (column 12)Add 3.5 μL HSET/Microtubule solution (3.3) (columns 1-11 & 13-24)Centrifuge at 1000 rpm for 1 min(pre-incubate enzyme and compound for 10 min)Add 1.5 μL of 10 μM ATP to start reaction gives a final [HSET] of 5 nM, [ATP] of 3 μM and [microtubule] of 70 μg/mL, centrifuge at 1000 rpm for 1 min and incubated at rt for 80 min.After the 80-minute incubation, stop reaction by adding 5 μL ADP-Glo reagent to all wells. Centrifuge for 1 min at 1000 rpm, leave for 40 min at rt.In the dark/away from direct light, add 10 μL Kinase Detection Reagent (KDR) to all wells, Seal plate with a Topseal (Perkin Elmer Cat #6050185) and centrifuge as above, leave for 40 min at rt covered in foil. |
| Affinity data for this assay | |
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