Cell Signaling and Analysis

Transcription

1 Cell ignaling and Analysis -3/7 Caspase-Glo TM 3/7 Assay Caspase-Glo TM 3/7 Assay 25 G , 96 1 G , 1 G , Caspase-Glo TM 3/7 Buffer Caspase-Glo TM 3/7 ubstrate (lyophilized) Caspase-Glo TM 3/7 Assay -3/7-3/7 caspase-3/7 DEVD 1Caspase-Glo TM Reagent /7 // Caspase-Glo TM 3/7 Assay / -- 1 / / 1 Z' 3-2 H Z-DEVD- Caspase-3/7 Z-DEVD- + - H 2 Luciferase Mg 2+ Light + ATP + 2-3/7DEVD - A. Luminescence (RLU, Blank ubtracted) B. Luminescence (RLU, Blank ubtracted) 1,6 1 1,4 1,2 1, , 1, , 2, 3, 4, 5, 6, Cell # Anti-Fas Treated Cells Untreated Cells.5 hour read 1 hour read 2 hour read 3 hour read 7, 8, 9, 1, 461MB4_3A Protocol o. TB , 1, Cell # 498MA4_3A Jurkat Jurkat -Fas mab 4.5A. Caspase-Glo TM 3/7 Reagent 1 B. Caspase-Glo TM 3/7 Reagent.5, 1, 2, 3 Prometech Journal 23 ew Product Guide 2

2 Cell ignaling and Analysis CellTiter-Blue TM Cell Viability Assay CellTiter-Blue TM Cell Viability Assay 2ml G , 2ml 961 1ml G , 1 1ml G , CellTiter-Blue TM Reagent CellTiter-Blue TM Cell Viability Assay 1 RFU (56/59nm) 3 CellTiter-Blue TM Cells/well (x1,) CellTiter-Blue TM Z' factor Z' 2 Apo-E Assay Fluorescence (56/59nm) 6, 5, 4, 3, 2, 1, HepG2 Jurkat Cells/Well (x 1 3 ) 3886MA11_2A -2 Protocol o. TB317 2JarkatHepG21µl/ CellTiter-Blue TM Reagent 2µl/ 1Labsystems Fluoroskan Ascent plate reader 56(2)EX/59(1)Em a + Resazurin Viable Cell Reduction Reactions a + Resorufin Emits fluorescence at 59nm CellTiter-Blue TM Cell Viability Assay 3889MA11_2A Fluorescence (56/59nm) 2,5 2, 1,5 1, Caspase Activity Resazurin Reduction CellTiter-Blue Assay Apo-E Assay ,2 1,6 2, taurosporine (nm) 6, 4, 2, 2 Jarkat1µl/5 CellTiter-Blue TM Reagent (2µl/) 5 (56Ex/59Em) Apo-E Homogeneous Caspase 3/7 Assay Reagent 12µl 1(485Ex/527Em) Fluorescence (485/527nm) 3884MA11_2A 3 Prometech Journal 23 ew Product Guide

3 Cell ignaling and Analysis on-ri Kinase-Glo TM Luminescent Kinase Assay Kinase-Glo TM Luminescent Kinase Assay 1ml V , 1ml ml V , 1ml V , 1 1ml V , Kinase-Glo TM Buffer Kinase-Glo TM ubstrate (lyophilized) Kinase-Glo TM Luminescent Kinase Assay ATP on-ri (HT) 1 (Kinase-Glo TM Reagent) ATP Z' (>.8) IC Z' >.8 UltraGlo TM Luciferase Protocol o. RLU 4, 35, 3, 25, 2, 15, 1, 5,.25u/well PKA o PKA Time (hours) 3892MA11_2A TB318 96Kinase-Glo TM Reagent (n=32).25 units/well PKA ( ) PKA ( ) 425% 3, 25, 1,, 1,, RLU 2, 15, RLU 1, 1, 1, 5, 1, ATP (µm) ATP 3893MA11_2A Prometech Journal 23 ew Product Guide Well # PKA LPAC library (igma-rbi) D 3896TA11_2A 4

4 Cell ignaling and Analysis on-ri ProFluor TM PKA Assay* ProFluor TM PKA Assay 4 V , 96 8 V , PKA R11 ubstrate, 1mM ratp, 1mM Protease Reagent tabilizer Reagent 5X Reaction Buffer A 5X Termination Buffer A ProFluor TM PKA Assay A PKA bisamide rhodamine 11 termination buffer PKA rhodamine Z'.8 PKA 1 Z' on-ri -2 onphosphorylated Phosphorylated 1, 9, 8, 7, 6, 5, 4, 3, 2, 1, 18, 16, 14, 12, 1, 8, 6, 4, Percent Phosphorylated Peptide ProFluor TM 2, Well # PKA LPAC library (igma-rbi) 6 72 ProFluor TM ATP 3 D 72 # 6 HA % 18 H-7 5.5% 3 H-8 2.6% 42 H % 59 U % 7 GW % 3878TA11_2A + Protease + Protease Protocol o. TB315 5 Prometech Fluorescent ProFluor TM onfluorescent * Patent Pending. This product is covered by U.. Pat. os. 4,557,862 and 4,64,893 and is sold for research use only. All other uses, including but not limited to use as a clinical diagnostic or therapeutic, require a separate license. Please contact Promega Corporatio Journal 23 ew Product Guide

5 Cell ignaling and Analysis on-ri ProFluor TM er/thr Phosphatase Assay ProFluor TM er/thr Phosphatase Assay 4 V , 96 8 V , /T PPase R11 ubstrate, 1mM Control AMC ubstrate, 1mM Protease Reagent MgCl2, 1M MnCl2, 2mM CaCl2, 1mM icl2, 1mM tabilizer Reagent 5X Reaction Buffer B 5X Termination Buffer B ProFluor TM er/thr Phosphatase Assay* / - 1 (PP1), PP2A, PP2BPP2C 11 (/T PPase R11 ubstrate) Control AMC ubstrate Termination Buffer 11/T PPase R11 ubstrate R Z' factor.8 Z' AAF-AMC Protocol o. 16, 14, 12, 1, o Phosphatase Protease Reagent 8, 6, 4, 2, onfluorescent /T Phosphatase Protease Reagent Fluorescent TB324 ProFluor TM er/thr Phosphatase Assay r 2 = Percent onphosphorylated Peptide ProFluor TM er/thr Phosphatase Assay 12, 1, 8, 6, 4, 2, Well # 384Z' 384Z' 3 D 6.25milliunit/ PP1 (Calbiochem, Cat# ) Z' TA6_3A Prometech Journal 23 ew Product Guide 6

6 Cell ignaling and Analysis P45 P45-Glo TM Assay* P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , P45-Glo TM Assay 1ml V , 5ml V , 96 1ml 25ml 115µl 384 1ml 45ml 2 125µl Modified Detection Reagent (lyophilized) P45-Glo TM Buffer P45-Glo TM Assay P45 P45 / P45 P45-Glo TM Assay P45 P45 P45 P45-Glo TM Assay P HPLCP45 & - 2 : P45-Glo <.16%5 P P45-Glo ubstrate a. b. c. d. 6 methyl ether (-ME) Cl H 3 C cytochrome P45 cytochrome P45 Reaction 6 chloroethyl ether (-CEE) 6 Deoxyluciferin (-H) 6 benzyl ether (-BE) Firefly Luciferase H Luciferase Reaction Light a. Demethylation b. Dechloroethylation c. Hydroxylation d. Debenzylation P45-Glo Assay P45 P45 7 Prometech Journal 23 ew Product Guide

7 P45-Glo TM Assay Luminogenic Cytochrome P45 Assay Detection Reagent reconstituted Detection Reagent P45-Glo Buffer - 1 P45 P45 96 KP4 (Kmapp).5pmol 1mM 3µM -CEE 2.5pmol 1mM 1µM -ME pmol 1mM 2µM -CEE 3 1.pmol 5mM 15µM -ME 3.5pmol 25mM 1µM -H 3 1.pmol 2mM 5µM -BE 3 1.pmol 1mM 15µM -BE µM 2µg 1mM P45 P45 965µl 38425µl Luminometer 4154MA5_3A /-CEE 5, 3, 1, cytochrome P45 (pmoles) P45-Glo Assay /-CEE 6, 4, 2, cytochrome P45 (pmoles) A. -CEE B. -ME 18, 15, 12, 9, 6, 12, 1, 8, 6, 4, Adult hepatic isoforms Fetal hepatic isoform onhepatic isoform /-ME /-ME 8, 24, 6, 4, 2, cytochrome P45 (pmoles) /-BE /-BE 5, 2,, 4, 3, 2, 1, cytochrome P45 (pmoles) /-H 1,, 75, 5, 25, 18, 12, 6, cytochrome P45 (pmoles) 1,5, 1,, 5, cytochrome P45 (pmoles) C. 5, 4, 3, 2, 1, -BE D. 4, 3, 2, 1, -H -P45 P45-Glo 13 -P454P45-Glo TM P45 A. -CEE,, - 7B. -ME,,, -3C. -BE, D. -H 4156TA5_3A cytochrome P45 (pmoles) P45-Glo TM P45-Glo TM Assay P45 Km371 () 3 (,,,,, ) P TA5_3A Protocol o. * Patent Pending. TB325 Prometech Journal 23 ew Product Guide 8