FINDING THE PATHWAY TO ASSAY OPTIMIZATION IS EASY LANCE® Ultra Serine/Threonine and Tyrosine Kinase Assay Selection Guide LANCE Ultra Serine/Threonine Kinase Selection Guide LANCE® Ultra TR-FRET reagents comprise the widest portfolio of validated kinase assay offerings available for rapid, sensitive and robust screening of purified kinase targets in a biochemical format. • We provide S/B ratiometric data for each LANCE Ultra assay to guide you to the best performing solution for your assay. • Rapid assay optimization every time. • Our selection guides contain over 300 kinases from a variety of suppliers. How to use this guide: 1.Locate your kinase • In many cases, up to three commercial kinase vendors have been tested. • Many common aliases are shown in parenthesis. 2.Best performing ULight™ substrates are listed for each enzyme according to performance criteria where signal to background (S/B) ratios are greater than 5.0 • S/B ratios (Signal at 665 nm / minus ATP control at 665 nm) are indicated in parenthesis. • All S/B ratios were obtained at fixed experimental conditions unless otherwise noted (see page 10). If you cannot find your kinase of interest, please ask your PerkinElmer sales specialist, as our list continues to expand. Two kits are available for testing purposes: • KinaSelect Ser/Thr kit (5 x 250 data points, TRF0300-C) – 5 ULight-labeled SER/THR kinase specific substrates + 5 matching Europium-labeled Antiphospho-Antibodies • KinaSelect TK kit (1,000 data points, TRF0301-D) – 1 ULight-labeled kinase specific substrate + 1 matching Europium-labeled Anti-phospho-Antibody 3.Based on your substrate choice, find the corresponding Europium-labeled anti-phospho-antibody on page 11 (LANCE Ultra products used for this study) to identify the TR-FRET donor anti-phospho-antibody for your LANCE Ultra assay. 2 www.perkinelmer.com/kinaseconnection LANCE® Ultra Reagents for Ser/Thr Kinase Assays Up to three kinase substrates are listed based on signal to background (S/B) ratios obtained with commercially available enzymes. S/B ratios (Signal at 665 nm / minus ATP control at 665 nm) are indicated in parenthesis. Kinase ULight Substrate 1 ACTR2B (ACVR2B) ULight-Histone H3 (Thr3; 10.4) ULight-Topo IIa (7.2) ULight-Topo IIa (10.9) ULight-Histone H3 (Thr3; 9.4) ULight-Topo IIa (5.6) ULight-PLK (5.4) ULight-Topo IIa (22.9) ULight-Topo IIa (27.9) ULight-Topo IIa (22.9) ULight-Histone H3 (Thr3; 10.1) ULight-rpS6 (20.5) ULight-PLK (19.1) ULight-PKC (15.2) ULight-rpS6 (21.7) ULight-PLK (20.1) ULight-Crosstide (16.6) ULight-rpS6 (21.6) ULight-PLK (20.7) ULight-PKC (15.6) ULight-Topo IIa (20.6) ULight-Acetyl-CoA Carboxylase (30.5) ULight-PLK (15.1) ULight-CREBtide (11.9) ULight-Acetyl-CoA Carboxylase (29.9) ULight-CREBtide (16.0) ULight-PLK (11.5) ULight-Topo IIa (7.7) ULight-CREBtide (5.8) ULight-CREBtide (5.1) ULight-PLK (20.5) ULight-Histone H3 (Ser10; 13.0) ULight-rpS6 (21.0) ULight-PLK (17.6) ULight-CREBtide (12.7) ULight-Histone H3 (Ser10; 12.7) ULight-PLK (7.6) ULight-PLK (16.2) ULight-Histone H3 (Ser10; 12.1) ULight-Histone H3 (Ser10; 8.4) ULight-Topo IIa (18.7) ULight-Histone H3 (Thr3; 11.0) GST-MAP2K1/ ULight anti-GST (9.3) GST-MAP2K1/ ULight anti-GST (9.6) ULight-CDC25 (8.4) ULight-Acetyl-CoA Carboxylase (7.9) ULight-CREBtide (16.6) ULight-CREBtide (18.6) ULight-CREBtide (17.2) ULight-rpS6 (22.3) ULight-PKC (16.1) ULight-CREBtide (15.5) ULight-rpS6 (23.6) ULight-CREBtide (20.8) ULight-mTOR (12.0) ULight-rpS6 (25.4) ULight-CREBtide (20.5) ULight-PLK (14.0) ULight-rpS6 (18.2) ULight-PKC (15.6) ULight-Crosstide (15.3) ULight-CREBtide (14.6) ULight-mTOR (8.6) ULight-PLK (6.0) ACV-R1 ACVR2A ACVRL1 (ALK1) BARK1 (ADRBK1, GRK2) ADRBK2 (BARK2, GRK3) BARK2 (ADRBK2, GRK3) AKT1 AKT2 AKT3 ALK4 (ACVR1B) AMPKa1/b1/g1 (PRKAA1/B1/G1) AMPKa2/b1/g1(PRKAA2/B1/G1) ARK5 (NuaK1) ARK5 (NuaK1) AurA (Aurora-A) AurB (AURKB)/INCENP Aurora-B AurC (AURKC) Aurora-C BMPR1B BMPR2 BRAF-wt BRAF [VE] BRSK2 CaMK1a (CAMK1)2 CaMK1b (PNCK)2 CaMK1d (CAMK1D)2 CaMK2a (CAMK2A)2 CaMK2b (CAMK2B)2 CaMK2d (CAMK2D)2 CaMK2g (CAMK2G)2 CaMK42 Newly added Kinases highlighted in Green 3 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences ProQinase Carna Biosciences Invitrogen Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 09-132 0372-0000-1 09-131 PV4883 01-111 PV3827 01-112 01-101 01-102 01-103 09-135 02-113 02-114 0300-0000-1 02-126 05-101 05-102 0190-0000-1 05-103 0315-0000-1 09-138 09-139 0403-0000-1 0404-0000-1 02-116 02-104 02-105 02-106 02-109 02-110 02-111 02-112 02-108 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 CAMKK12 CAMKK2 CAMKK22 CDC2/CycB1 CDC2L6/CycC CDC42BPB (MRCKb) MRCKb (CDC42BPB) CDC7/ASK CDK1/CycA CDK1/CycB1 CDK1/CycE CDK2/CycA CDK2/CycE1 CDK3/CycE1 CDK4/CycD1 CDK4/CycD3 CDK5/p25 CDK5/p25NCK CDK5/p35NCK CDK6/CycD1 CDK6/CycD3 CDK7/CycH/MAT1 CDK9/CycT1 CGK2 (PKG2, PRKG2) PRKG2 (CGK2, PKG2) CHK1 (CHEK1) CHK2 (CHEK2) CK1a (CSNK1A1) CK1-alpha1 CK1d (CSNK1D) CK1e (CSNK1E) CK1g1 (CSNK1G1) CK1g2 (CSNK1G2) CK1g3 (CSNK1G3) CK2-alpha1 CK2a1/b (CSNK2A1/B) CK2-alpha2 CK2a2/b (CSNK2A2/B) CLK1 ULight-p70S6K (25.1) ULight-Histone H3 (Thr3; 12.2) ULight-Histone H3 (Thr3; 12.0) ULight-p70S6K (5.5) ULight-MBP (43.2) ULight-4E-BP1 (37.2) ULight-Histone H3 (Thr3; 10.0) ULight-Histone H3 (Ser10; 6.7) ULight-Histone H3 (Ser10; 6.9) ULight-Topo IIa (14.6) ULight-4E-BP1 (37.9) ULight-MBP (21.9) ULight-4E-BP1 (39.6) ULight-MBP (27.8) ULight-4E-BP1 (39.2) ULight-MBP (21.5) ULight-MBP (39.9) ULight-4E-BP1 (36.7) ULight-4E-BP1 (33.5) ULight-MBP (28.5) ULight-MBP (38.9) ULight-4E-BP1 (23.3) ULight-4E-BP1 (38.7) ULight-MBP (21.6) ULight-Histone H3 (Thr3; 6.3) ULight-MBP (37.2) ULight-4E-BP1 (32.3) ULight-MBP (37.1) ULight-4E-BP1 (36.5) ULight-4E-BP1 (34.2) ULight-MBP (24.9) ULight-4E-BP1 (40.3) ULight-MBP (22.9) ULight-Histone H3 (Thr3; 7.0) ULight-4E-BP1 (39.6) ULight-MBP (17.0) ULight-MBP (35.7) ULight-4E-BP1 (35.4) ULight p70S6K (5.9) ULight-MBP (19.0) ULight-4E-BP1 (28.1) ULight-MBP (14.4) ULight-PLK (14.1) ULight-CREBtide (11.1) ULight-Histone H3 (Thr3; 7.1) ULight-PLK (15.7) ULight-CREB (12.9) ULight-rpS6 (5.5) ULight-PKC (15.7) ULight-CREBtide (11.2) ULight-Acetyl-CoA Carboxylase (8.1) ULight-CREBtide (14.1) ULight-PLK (7.3) ULight-rpS6 (6.4) ULight-Topo IIa (13.7) ULight-Histone H3 (Thr3; 5.8) ULight-Topo IIa (15.0) ULight-Histone H3 (Thr3; 5.7) ULight-Topo IIa (23.5) ULight-Topo IIa (23.7) ULight-Histone H3 (Thr3; 10.2) ULight-Histone H3 (Thr3; 14.6) ULight-Histone H3 (Thr3; 10.1) ULight-Topo IIa (7.2) ULight-Histone H3 (Thr3; 12.9) ULight-Topo IIa (22.0) ULight-Histone H3 (Thr3; 6.8) ULight-IkBa (28.7) ULight-Topo IIa (13.8) ULight-Histone H3 (Thr3; 10.9) ULight-Topo IIa (21.4) ULight-IkBa (29.6) ULight-Topo IIa (14.6) ULight-Histone H3 (Thr3; 10.1) ULight-MBP (41.2) ULight-4E-BP1 (25.7) ULight-Histone H3 (Ser10; 8.5) Newly added Kinases highlighted in Green 4 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase ProQinase ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase ProQinase ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences 05-107 05-108 05-108 04-102 04-112 0382-0000-1 01-108 05-109 0134-0054-1 0134-0135-1 0134-0055-1 04-103 04-165 04-104 0142-0143-1 04-105 04-106 0356-0389-1 0356-0355-1 0051-0143-1 04-107 04-108 04-110 01-143 PV3973 02-117 02-162 03-101 0406-0000-1 03-103 03-104 03-105 03-106 03-107 0124-0000-1 05-184 0412-0000-1 05-185 04-126 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase CLK2 CLK3 COT COT (MAP3K8) COT (MAP3K8) CRIK (CIT) DAPK1 DAPK1 DAPK2 DAPK3 DCAMKL1 DCAMKL2 DLK (MAP3K12) DNA PK3 DRAK1 (STK17A) DYRK1A DYRK1B DYRK2 DYRK3 DYRK4 Erk1 (MAPK3) Erk2 (MAPK1) ERK2 Erk5 (MAPK7) Erk7 (MAPK15) GPRK4 (GRK4) GPRK6 (GRK6) GPRK7 (GRK7) GRK5 GSK3a (GSK3A) GSK3b (GSK3B) Haspin (GSG2) HGK (MAP4K4) HIPK1 HIPK2 HIPK3 HIPK4 HRI IKKa (CHUK) ULight Substrate 1 Newly added Kinases highlighted in Green 5 ULight Substrate 2 ULight Substrate 3 ULight-MBP (11.9) ULight-CREBtide (9.0) ULight-Histone H3 (Ser10; 8.3) ULight-rpS6 (8.5) ULight-MBP (8.5) ULight-Histone H3 (Ser10; 7.2) GST-MAP2K1 / ULight-anti-GST (Mn2+; 8.0) GST-MAP2K1 / ULight-anti-GST (6.6) ULight-p70S6K (Mn2+; 5.3) GST-MAP2K1 / ULight-anti-GST (8.6) ULight-p70S6K (Mn+2; 11.0) ULight-Acetyl-CoA Carboxylase (Mn+2; 7.8) GST-MAP2K1 / ULight-anti-GST (Mn2+; 7.8) ULight-Histone H3 (Ser10; 8.3) ULight-PLK (18.1) ULight-Histone H3 (Ser10; 9.3) ULight-Histone H3 (Thr3; 8.8) ULight-Histone H3 (Thr3; 9.8) ULight-PLK (6.4) ULight-Histone H3 (Thr3; 6.6) ULight-Histone H3 (Ser10; 6.3) ULight-PLK (6.1) ULight-CREBtide (7.2) ULight-CREBtide (9.3) ULight-PKC (11.6) ULight-CREBtide (11.3) ULight-Histone H3 (Thr3; 10.9) ULight-mTOR (40 U; S/B +/- DNA: 6.6) ULight-Histone H3 (Thr3; 6.2) ULight-MBP (18.5) ULight-4E-BP1 (7.4) ULight-4E-BP1 (24.6) ULight-MBP (23.2) ULight-MBP (33.4) ULight-4E-BP1 (30.0) ULight-Histone H3 (Thr3; 9.5) ULight-4E-BP1 (39.9) ULight-MBP (22.0) ULight-Histone H3 (Thr3; 7.0) ULight-4E-BP1 (36.3) ULight-MBP (26.7) ULight-Histone H3 (Thr3; 10.1) ULight-4E-BP1 (37.9) GST-ATF-2 / ULight-anti-GST (37.6) ULight-MBP (26.6) ULight-4E-BP1 (37.1) GST-ATF-2 / ULight-anti-GST (35.1) ULight-MBP (28.0) GST-ATF-2 / ULight-anti-GST (40.0) ULight-4E-BP1 (31.9) ULight-MBP (19.4) ULight-4E-BP1 (22.4) ULight MBP (5.5) ULight-4E-BP1 (22.3) ULight-Topo IIa (18.8) ULight-p70S6K (14.2) ULight-Histone H3 (Thr3; 11.6) ULight-Histone H3 (Thr3; 10.8) ULight-Histone H3 (Thr3; 10.2) ULight-Topo IIa (5.9) ULight-IkBa (26.5) ULight-Topo IIa (26.3) ULight-Histone H3 (Thr3; 11.7) ULight-4E-BP1 (34.5) ULight-Histone H3 (Thr3; 8.0) ULight MBP (5.1) ULight-4E-BP1 (35.9) ULight-IkBa (13.3) ULight-Histone H3 (Thr3; 15.5) ULight MBP (5.5) ULight-p70S6K (23.8) ULight-Histone H3 (Thr3; 6.3) ULight-4E-BP1 (30.8) ULight-MBP (22.9) ULight-Histone H3 (Thr3; 5.3) ULight-MBP (37.4) ULight-4E-BP1 (36.9) ULight-MBP (22.9) ULight-4E-BP1 (37.7) ULight-MBP (21.2) ULight-Histone H3 (Thr3; 7.1) ULight-PLK (6.2) ULight-IkBa (22.4) ULight-rpS6 (17.9) ULight-Acetyl-CoA Carboxylase (11.8) Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Promega Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences 04-127 04-128 0180-0000-1 07-301 07-301 01-104 02-134 0332-0000-1 PV3614 02-136 02-139 02-140 09-111 V7870 02-137 04-130 04-131 04-132 04-133 PV3871 04-142 04-143 0634-0000-7 04-146 04-147 01-313 01-315 01-116 PV3824 04-140 04-141 05-111 07-137 04-135 04-136 04-137 04-138 0444-0000-1 05-112 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 IKKb IKK-beta IKKe (IKBKE) IKK-epsilon IRAK1 IRAK4 IRAK4 IRAK4 IRAK4 JNK1 (MAPK8) JNK2 (MAPK9) JNK3 (MAPK10) LRRK2 LRRK2 [G2019S] MAP2K4 MAP2K6 (MKK6) MAP3K1 MAP4K2 MAP4K5 (KHS1) MAPKAPK2 MAPKAPK3 MAPKAPK3 MARK1 MARK1 MARK2 (EMK1) MARK3 (C-TAK1) MARK3 (C-TAK1) MARK4 (MARKL1) MELK MGC42105 (B3KVM1) MINK (MINK1) MLK1 (MAP3K9) MLK2 (MAP3K10) MLK3 (MAP3K11) MNK1 (MKNK1) MNK2 (MKNK2) MOS MRCKa (CDC42BPA) ULight-p70S6K (30.4) ULight-IkBa (25.6) ULight-rpS6 (22.1) ULight-IkBa (25.5) ULight-Histone H3 (Thr3; 5.8) ULight-p70S6K (22.1) ULight-rpS6 (20.2) ULight-PLK (15.8) ULight-rpS6 (22.0) ULight-p70S6K (15.5) ULight-PLK (12.5) ULight-p70S6K (28.3) ULight-Acetyl-CoA Carboxylase (17.7) ULight-Histone H3 (Thr3; 14.4) ULight-Acetyl-CoA Carboxylase (31.9) ULight-Histone H3 (Thr3; 9.9) ULight-p70S6K (8.6) ULight-p70S6K (Mn+2; 32.4) ULight-Acetyl-CoA Carboxylase (Mn+2; 30.6) ULight-Histone H3 (Thr3; Mn+2; 8.3) ULight-Acetyl-CoA Carboxylase (23.9) ULight-Histone H3 (Thr3; 12.3) ULight-p70S6K (Mn+2; 28.1) ULight-Acetyl-CoA Carboxylase (Mn+2; 24.9) ULight-Histone H3 (Thr3; Mn+2; 10.0) ULight-4E-BP1 (34.5) GST-ATF-2 / ULight-anti-GST (32.0) ULight-MBP (21.4) ULight-4E-BP1 (35.0) GST-ATF-2 / ULight-anti-GST (32.8) ULight-MBP (20.8) GST-ATF-2 / ULight-anti-GST (33.7) ULight-4E-BP1 (32.8) ULight-MBP (19.8) ULight-PLK (19.9) ULight-CREBtide (15.4) ULight-Histone H3 (Thr3; 10.4) ULight-Histone H3 (Thr3; 15.4) GST-p38a / ULight-anti-GST (22.1) GST-p38a/ ULight-anti-GST (23.1) ULight-Acetyl-CoA Carboxylase (5.3) ULight-rpS6 (18.3) ULight-p70S6K (9.2) ULight-p70S6K (35.6) ULight-rpS6 (24.9) ULight-Acetyl-CoA Carboxylase (23.8) ULight-4E-BP1 (32.2) ULight-MBP (31.4) ULight-rpS6 (13.2) ULight-CREBtide (13.0) ULight-PKC (12.3) ULight-mTOR (11.8) ULight-CREBtide (11.3) ULight-CDC25 (8.2) ULight-PLK (18.1) ULight-Acetyl-CoA Carboxylase (13.9) ULight-Acetyl-CoA Carboxylase (9.4) ULight-PLK (9.3) ULight-CDC25 (6.7) ULight-PLK (18.0) ULight-Acetyl-CoA Carboxylase (15.6) ULight-CDC25 (6.9) ULight-PLK (12.3) ULight-Acetyl-CoA Carboxylase (11.9) ULight-CDC25 (7.6) ULight-Acetyl-CoA Carboxylase (15.1) ULight-PLK (13.2) ULight-CDC25 (7.1) ULight-Acetyl-CoA Carboxylase (25.7) ULight-PLK (18.8) ULight-PKC (6.5) ULight-CREBtide (13.9) ULight-PLK (10.8) ULight-CDC25 (7.4) ULight-CREBtide (7.2) ULight-p70S6K (55.1) ULight-PKC (14.5) ULight-rpS6 (11.8) GST-MAP2K1/ULight-anti-GST (11.4) ULight-Histone H3 (Thr3; 11.0) GST-MAP2K1/ULight-anti-GST (6.4) ULight-CREBtide (10.4) ULight-Histone H3 (Ser10; 9.9) ULight-rpS6 (18.9) ULight-CREBtide (13.6) ULight-Histone H3 (Ser10; 13.2) ULight-Topo IIa (9.1) GST-MAP2K1/ULight-anti-GST (6.3) ULight-Histone H3 (Ser10; 7.5) Newly added Kinases highlighted in Green 6 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 05-084 0258-0000-1 05-114 0320-0000-1 09-101 09-145 09-145 0586-0000-1 0586-0000-1 04-163 04-164 04-150 09-110 PV4881 07-044 07-046 07-103 07-111 PV3682 02-142 02-143 0633-0000-1 02-120 0432-0000-1 02-121 02-122 0433-0000-1 02-123 02-124 02-125 07-139 09-015 09-116 2109-017 02-145 02-146 05-118 01-107 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 MSK1 (RPS6KA5) MSK2 (RPS6KA4) MST1 (STK4) MST1 (STK4) MST1 (STK4) MST2 (STK3) MST2 (STK3) MST3 (STK24) MST4 (MASK) MST4 mTOR (1362-end) NDR1 (STK38) NEK1 NEK2 NEK2 NEK3 NEK4 NEK6 NEK7 NEK9 NEK11 NLK Nuak2 (SNARK) Nuak2 (SNARK) OSR1 (OXSR1) p38a (MAPK14) p38-alpha (MAPK14) p38b (MAPK11) p38-beta (MAPK11) p38d (MAPK13) p38g (MAPK12) p70S6K (S6K; RPS6KB1) S6K (p70S6K, RPS6KB1) p70 S6 Kinase (T412E) PAK1 PAK1 PAK2 PAK2 PAK2 ULight-rpS6 (26.4) ULight-PLK (20.6) ULight-CREBtide (14.5) ULight-rpS6 (25.0) ULight-PLK (19.8) ULight-Crosstide (16.9) ULight-p70S6K (13.3) ULight PKC (12.8) ULight-Histone H3 (Thr3; 8.9) ULight PKC (11.9) ULight-Histone H3 (Thr3; 8.4) ULight-p70S6K (6.5) ULight PKC (14.0) ULight-Histone H3 (Thr3; 6.5) ULight-rpS6 (5.5) ULight-Histone H3 (Thr3; 8.0) ULight PKC (5.2) ULight-p70S6K (12.5) ULight PKC (9.0) ULight-Histone H3 (Thr3; 6.2) ULight PKC (9.3) ULight-rpS6 (8.9) ULight PKC (14.6) ULight-PKC (16.3) ULight-rpS6 (8.5) ULight-4E-BP1 (Mn2+; 36.9) ULight-p70S6K (Mn2+; 18.3) ULight-Histone H3 (Ser10; 8.7) ULight-p70S6K (60.0) ULight-Acetyl-CoA Carboxylase (24.0) ULight-rpS6 (18.6) ULight-p70S6K (43.3) ULight-rpS6 (18.2) ULight-PKC (15.5) ULight-p70S6K (30.7) ULight-rpS6 (14.1) ULight-CREB (10.9) ULight-p70S6K (26.9) ULight-4E-BP1 (18.2) ULight-Histone H3 (Thr3; 10.1) ULight-p70S6K (40.1) ULight-Histone H3 (Thr3; 13.6) ULight-rpS6 (6.1) ULight-p70S6K (32.0) ULight-p70S6K (33.4) ULight-p70S6K (48.9) ULight-rpS6 (16.5) ULight-Acetyl-CoA Carboxylase (13.0) ULight-p70S6K (20.0) ULight-Histone H3 (Thr3; 14.9) ULight-PLK (6.0) ULight-Histone H3 (Thr3; 7.2) ULight-Histone H3 (Thr3; 5.8) ULight-PLK (5.5) ULight-Histone H3 (Thr3; 5.6) ULight-4E-BP1 (37.0) GST-ATF-2 / ULight-anti-GST (32.1) ULight-MBP (27.9) GST-ATF-2 / ULight-anti-GST (36.7) ULight-4E-BP1 (33.1) ULight-MBP (5.4) ULight-MBP (40.6) GST-ATF-2 / ULight-anti-GST (36.9) ULight-4E-BP1 (32.6) GST-ATF-2 / ULight-anti-GST (36.8) ULight-4E-BP1 (35.8) ULight-MBP (14.2) ULight-MBP (41.2) GST-ATF-2 / ULight-anti-GST (35.1) ULight-4E-BP1 (30.7) ULight-4E-BP1 (37.7) GST-ATF-2 / ULight-anti-GST (37.0) ULight-MBP (33.6) ULight-rpS6 (18.1) ULight-CREBtide (15.0) ULight-Histone H3 (Ser10; 9.4) ULight-rpS6 (16.0) ULight-mTOR (11.8) ULight-mTOR (11.6) ULight-Crosstide (8.1) ULight-PLK (12.3) ULight-PLK (15.6) ULight-PLK (18.7) ULight-Histone H3 (Ser10; 10.5) ULight-CREBtide (8.3) ULight-PLK (16.7) ULight-PLK (13.2) Newly added Kinases highlighted in Green 7 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences Millipore Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Millipore Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Millipore Carna Biosciences ProQinase Millipore Carna Biosciences ProQinase 01-147 01-148 14-624 07-116 0667-0000-1 07-117 0699-0000-1 07-118 07-119 0286-0000-1 14-770 01-125 05-123 05-226 0290-0000-1 05-127 05-128 05-130 05-131 05-133 05-125 0253-0000-1 02-127 0414-0000-1 07-122 04-152 0443-0000-3 04-153 0455-0000-3 04-154 04-155 01-154 0318-0000-2 14-486 07-123 0357-0000-1 14-481 07-124 0304-0000-1 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 PAK3 PAK3 PAK5 (PAK7) PAK61 PAK6 PASK PBK1 PEK (EIF2AK3) PGK (PRKG1) PRKG1 (PGK) PHKG1 PHKG2 PIM1 PIM1 PIM2 PIM2 PIM3 PKACa (PRKACA) PKACb (PRKACB) PKCa (PRKCA) PKCb1 (PRKCB1) PKC-beta1 PKCb2 (PRKCB2) PKC-beta2 (PRKCB2) PKCd (PRKCD) PKC-delta PKCe (PRKCE) PKC-epsilon PKCg (PRKCG) PKC-gamma (PRKCG) PKCh (PRKCH) PKC-eta (PRKCH) PKCi (PRKCI) PKC-iota PKCu (PRKCQ) PKC-theta PKCz (PRKCZ) PKC-zeta PKC-mu (PKD-1, PRKD1) ULight-PLK (19.9) ULight-Histone H3 (Ser10; 12.2) ULight-CREBtide (8.2) ULight-PLK (19.0) ULight-PLK (18.5) ULight-PLK (5h; 7.0) ULight-Histone H3 (Thr3; 8.5) ULight-PLK (5.5) ULight-CREBtide (12.8) ULight-Crosstide (12.6) ULight-mTOR (10.4) ULight-Acetyl-CoA Carboxylase (5 h; 7.1) ULight-p70S6K (51.9) ULight-Topo IIa (18.0) ULight-Acetyl-CoA Carboxylase (15.4) ULight-CREBtide (11.2) ULight-PLK (11.1) ULight-Histone H3 (Thr3; 10.6) ULight-CREBtide (13.8) ULight-PLK (12.5) ULight-CREBtide (13.0) ULight-Histone H3 (Thr3; 8.5) ULight-rpS6 (7.0) ULight-rpS6 (15.6) ULight-CREBtide (14.1) ULight-mTOR (9.7) ULight-CREBtide (8.5) ULight-mTOR (8.5) ULight-rpS6 (21.5) ULight-mTOR (12.6) ULight-CREBtide (11.0) ULight-rpS6 (20.9) ULight-CREBtide (13.3) ULight-mTOR (11.3) ULight-rpS6 (10.4) ULight-mTOR (6.6) ULight-Histone H3 (Thr3; 6.4) ULight-rpS6 (24.7) ULight-CREBtide (18.6) ULight-mTOR (11.7) ULight-rpS6 (16.8) ULight-PLK (16.6) ULight-CROSStide (16.6) ULight-rpS6 (23.7) ULight-PLK (20.8) ULight-CREBtide (18.2) ULight-rpS6 (19.4) ULight-PKC (14.5) ULight-CREBtide (11.7) ULight-PKC (15.6) ULight-CREBtide (13.0) ULight-Histone H3 (Thr3; 6.4) ULight-PKC (15.5) ULight-CREB (10.8) ULight-PLK (6.3) ULight-rpS6 (19.1) ULight-PKC (14.0) ULight-CREBtide (12.5) ULight-PKC (16.2) ULight-CREBtide (11.1) ULight-PKC (14.8) ULight-CREBtide (13.4) ULight-PKC (14.3) ULight-CREB (10.9) ULight-rpS6 (8.6) ULight-PKC (14.9) ULight-CREBtide (13.5) ULight-PKC (13.0) ULight-CREB (9.3) ULight-PLK (5.7) ULight-PKC (15.6) ULight-CREBtide (13.7) ULight-rpS6 (9.1) ULight-PKC (13.5) ULight-rpS6 (10.2) ULight-CREBtide (9.9) ULight-CREBtide (14.9) ULight-PKC (14.3) ULight-rpS6 (9.5) ULight-rpS6 (22.4) ULight-PLK (13.2) ULight-PKC (10.6) ULight-PKC (15.4) ULight-CREBtide (14.2) ULight-rpS6 (8.9) ULight-PKC (15.0) ULight-CREB (10.8) ULight-PKC (15.1) ULight-CREBtide (13.5) ULight-rpS6 (12.3) ULight-PKC (14.5) ULight-CREB (10.4) ULight-rpS6 (5.5) ULight-PKC (14.1) ULight-CREBtide (14.0) ULight-rpS6 (12.9) ULight-PKC (14.1) ULight-CREBtide (8.5) ULight-PLK (5.6) ULight-CREBtide (10.2) ULight-PLK (8.4) ULight-PKC (5.6) Newly added Kinases highlighted in Green 8 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase ProQinase 07-125 042-0000-1 07-127 07-128 0423-0000-1 02-128 05-168 05-155 01-142 PV4340 02-152 02-153 02-054 0186-0000-1 02-155 0223-0000-1 02-156 01-127 01-128 01-133 01-134 0067-0000-1 01-165 0116-0000-1 01-135 0232-0000-1 01-136 0203-0000-1 01-137 0204-0000-1 01-138) 0420-0000-1 01-139 0114-0000-1 01-140 0113-0000-1 01-141 0117-0000-1 0115-0000-1 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 PKD1 (PRKD1, PKC-mu) PKC-nu (PRKD3, EPK2) PKD3 (PRKD3, PKC-nu) PKD2 (PRKD2) PKN1 (PRK1) PRK1 (PKN1) PKN2 PKR (EIF2AK2) PLK1 PLK1 PLK2 (SNK) PLK2 (SNK) PLK3 PLK3 PRKX QIK (SNF1LK2) SNF1LK2 (QIK) RIPK5 ROCK1 ROCK2 RSK1 (RPS6KA1) RSK2 (RPS6KA3) RSK3 (RPS6KA2) RSK4 (RPS6KA6) SGK SGK2 SGK3 (SGKL) SGK3 SIK SLK skMLCK (MYLK2)1-2 SRPK2 STK33 TAK1-TAB1 (MAP3K7) TAOK2 TAOK3 TBK1 ULight-PLK (14.4) ULight-CREBtide (6.0) ULight-CREBtide (12.5) ULight-PLK (5.4) ULight-CREBtide (14.4) ULight-Histone H3 (Thr3; 8.8) ULight-PLK (6.0) ULight-CREBtide (14.1) ULight-PLK (10.3) ULight-CREBtide (14.1) ULight-PKC (10.5) ULight-rpS6 (7.5) ULight-rpS6 (17.9) ULight-PKC (13.0) ULight-CREBtide (11.0) ULight-CREBtide (11.9) ULight-Histone H3 (Thr3; 10.8) ULight-p70S6K (24.8) ULight-Topo IIa (23.0) ULight-Histone H3 (Thr3; 13.4) ULight-Topo IIa (7.0) ULight-Histone H3 (Thr3; 5.7) ULight-Topo IIa (22.9) ULight-Histone H3 (Thr3; 6.4) ULight-Topo IIa (22.6) ULight-PLK (8.7) ULight-IkBa (27.5) ULight-Topo IIa (20.4) ULight-4E-BP1 (15.0) ULight-Topo IIa (18.6) ULight-Histone H3 (Thr3; 6.1) ULight-4E-BP1 (5.1) ULight-PLK (19.7) ULight-CREBtide (13.8) ULight-PKC (13.7) ULight-Acetyl-CoA Carboxylase (28.8) ULight-CREBtide (15.1) ULight-PLK (14.1) ULight-Acetyl-CoA Carboxylase (24.8) ULight-PLK (14.5) ULight-CREBtide (9.3) ULight-p70S6K (5.7) ULight-rpS6 (23.4) ULight-PLK (19.6) ULight-PKC (11.3) ULight-rpS6 (20.0) ULight-PLK (16.9) ULight-CREBtide (10.4) ULight-rpS6 (22.0) ULight-PLK (20.5) ULight-CREBtide (16.0) ULight-rpS6 (20.9) ULight-Crosstide (18.8) ULight-PLK (16.7) ULight-rpS6 (25.9) ULight-PLK (18.5) ULight-CREBtide (16.2) ULight-rpS6 (23.8) ULight-CREBtide (22.1) ULight-PLK (18.4) ULight-rpS6 (20.8) ULight-PLK (20.6) ULight-Crosstide (18.4) ULight-PLK (21.3) ULight-rpS6 (21.1) ULight-Crosstide (18.9) ULight-rpS6 (22.7) ULight-PLK (17.7) ULight-Crosstide (17.4) ULight-rpS6 (21.4) ULight-PLK (18.0) ULight-mTOR (12.7) ULight-rpS6 (11.6) ULight-CREBtide (5.8) ULight-PLK (5h; 15.5) ULight-CREBtide (5h; 12.9) ULight-Histone H3 (Thr3; 6.1) ULight-rpS6 (13.1) ULight-mTOR (9.5) ULight-Topo IIa (5.8) ULight-p70S6K (5.6) ULight-p70S6K (15.6) ULight-PKC (5.7) ULight-rpS6 (5.2) ULight-Histone H3 (Thr3; 9.3) ULight-p70S6K (7.9) ULight-p70S6K (34.5) ULight-rpS6 (24.8) ULight-Acetyl-CoA Carboxylase (24.5) Newly added Kinases highlighted in Green 9 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Invitrogen Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Millipore Carna Biosciences Carna Biosciences ProQinase Millipore Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 02-157 0661-0000-1 02-159 02-158 01-144 0207-0000-1 01-145 05-156 05-157 0183-0000-1 05-158 0277-0000-1 05-159 0437-0000-1 01-130 02-129 PV4792 PV4678 01-109 01-110 01-149 01-150 01-151 01-152 01-158 01-159 01-160 0198-0000-2 14-652 07-129 02-150 0381-0000-1 14-671 09-019 07-133 07-135 05-115 New LANCE Ultra assays highlighted in Blue www.perkinelmer.com/kinaseconnection Kinase ULight Substrate 1 TGFbR1 (TGFBR1) TLK1 TLK2 TSK2 TSSK1 TSSK2 TTBK1 TTBK2 VRK1 VRK2 WEE1 YSK1 (STK25) ZAK ULight-Topo IIa (21.3) ULight-Topo IIa (17.4) ULight-Histone H3 (Thr3; 15.2) ULight-p70S6K (9.4) ULight-Topo IIa (17.9) ULight-Histone H3 (Thr3; 17.2) ULight-p70S6K (13.8) ULight-Acetyl-CoA Carboxylase (17.9) ULight-PLK (16.2) ULight-Histone H3 (Thr 3;8.3) ULight-PLK (17.9) ULight-Acetyl-CoA Carboxylase (16.2) ULight-CDC25 (6.6) ULight-PLK (20.2) ULight-Acetyl-CoA Carboxylase (15.6) ULight-CREBtide (11.9) ULight-Topo IIa (24.9) ULight-Histone H3 (Thr3; 17.8) ULight-p70S6K (13.4) ULight-Topo IIa (24.2) ULight-Histone H3 (Thr3; 17.4) ULight-rpS6 (10.3) ULight-Histone H3 (Thr3; 8.7) ULight-Histone H3 (Thr3; 15.1) ULight-Histone H3 (Thr3; 6.4) ULight-rpS6 (14.3) ULight-p70S6K (5.7) ULight-p70S6K (36.1) ULight-Histone H3 (Thr3; 15.5) ULight-mTOR (11.4) Newly added Kinases highlighted in Green ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 09-141 05-166 05-167 0220-0000-1 02-364 02-165 03-108 03-109 0302-0000-1 03-111 05-177 07-136 09-120 New LANCE Ultra assays highlighted in Blue Kinase reaction time is 5h CaCl2 and Calmodulin at 1 mM and 10 µg/mL, respectively, are added to the kinase reactions 3 DNA PK: assay buffer and conditions as recommended by Promega. ATP: 200 μM final, kinase reaction incubation time 1 h Kinases were assayed in standard conditions, using reagent concentrations and kinase buffer described below. All modifications to standard assay conditions are indicated in the table. Kinase assay components: Kinase buffer (pH 7.5): Kinases: 20 nM (various suppliers) 50 mM HEPES ATP: 200 µM 10 mM MgCl2 ULight substrates in kinase reactions: 50 nM (100 nM for ULight-CDC25) 3 mM MnCl2 (when indicated) GST-protein substrates in kinase reactions: 30 nM (when indicated) 1 mM EGTA ULight-anti-GST antibody in assay: 30 nM (when indicated) 2 mM DTT and 0.01% Tween 20 Europium anti-phospho-substrate antibodies in assay: 2 nM After incubation at 23°C for 1 h (or 5 h when indicated), kinase reactions were stopped with EDTA. Antibodies were added and incubated for 1 h prior to detection with the EnVision. 1 2 NOTE: All kinases were sourced from commercial vendors from commercially available lots. In many cases, the same kinase was tested from multiple vendors. If the results obtained with kinases from different vendors were very different in our testing, we have shown all suppliers’ data. If the results were similar, we have shown only one representative set of test results. We encourage you to qualify your kinase activity to ensure that the assay signal is specific to the enzyme tested. 10 www.perkinelmer.com/kinaseconnection LANCE® Ultra products used for this study: Labeled Antibody ULight-CREBtide Europium-anti-phospho-CREB ULight-MBP Europium-anti-phospho-MBP ULight-Crosstide Europium-anti-phospho-Crosstide ULight-PLK Europium-anti-phospho-PLK ULight-PKC Europium anti-phospho-PKC ULight-IkBa Europium-anti-phospho-IkBa ULight-Histone H3 (Thr3/Ser10) Europium-anti-phospho-Histone H3 (Ser10) Europium-anti-phospho-Histone H3 (Thr3) ULight-mTOR Europium-anti phospho-mTOR ULight-p70S6K Europium-anti-phospho-p70S6K ULight-Acetyl-CoA Carboxylase Europium-anti-phospho-Acetyl-CoA Carboxylase ULight-Cdc25C Europium-anti-phospho-(Ser) 14-3-3 binding motif ULight-4E-BP1 Europium-anti-phospho-4E-BP1 ULight-rpS6 Europium-anti-phospho-rpS6 ULight-Topo IIa Europium-anti-phospho-Topo IIa ULight-anti-GST Europium-anti-phospho-ATF-2 Europium-anti-phospho-p38a Europium-anti-phospho-MEK1/2 11 Description Catalog No. Phospho-Motif ULight-CREBtide (Ser133) Europium-anti-phospho-CREB (Ser133) ULight-Myelin Basic Protein Peptide Europium-anti-phospho-Myelin Basic Protein ULight-Crosstide (GSK-3a Ser21) Europium-anti-phospho-Crosstide (GSK-3a Ser21) ULight-PLK (Ser137) Peptide Europium-anti-phospho-PLK (Ser137) ULight-PKC Substrate (Ala25Ser) Europium anti-phospho-PKC Peptide (Ala25Ser) ULight-IkappaB-alpha (Ser32/36) Peptide Europium-anti-phospho-IkappaB-alpha (Ser32/36) ULight-Histone H3 (Thr3/Ser10) Peptide Europium-anti-phospho-Histone H3 (Ser10) Europium-anti-phospho-Histone H3 (Thr3 ULight-mTOR (Ser2448) Peptide Europium-anti phospho-mTOR (Ser2448) ULight-p70S6K (Thr389) Peptide Europium-anti-phospho-p70S6K (Thr389) ULight-Acetyl-CoA Carboxylase (Ser79) SAMS Peptide Europium-anti-phospho-Acetyl-CoA Carboxylase (Ser79) ULight-Cdc25C (Ser216) Peptide Europium-anti-phospho-(Ser) 14-3-3 binding motif, mAb 4E2 ULight-eIF4E-binding protein 1 (Thr37/46) Peptide Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) ULight-40S Ribosomal Protein S6 (pSer235/Ser236) Peptide Europium-anti-phospho-40S Ribosomal Protein S6 (Ser235/236) ULight-DNA Topoisomerase 2-alpha (Thr1342) Peptide Europium-anti-phospho-DNA Topoisomerase 2-alpha (Thr1342) ULight-anti-GST Europium-anti-phospho-ATF-2 (Thr53) ATF-2 Fusion Protein Europium-anti-phospho-MAP Kinase p38-alpha (Thr180/Tyr182) GST-p38a (MAPK14), inactive Europium-anti-phospho-MEK1/2 (Ser217/221) GST-MAP2K1, inactive TRF0107 TRF0200 TRF0109 TRF0201 TRF0106 TRF0202 TRF0110 TRF0203 TRF0108 TRF0207 TRF0113 TRF0206 TRF0125 TRF0210 TRF0211 TRF0119 TRF0209 TRF0126 TRF0214 TRF0118 TRF0208 TRF0123 AD0192 TRF0128 TRF0216 TRF0129 TRF0217 TRF0130 TRF0218 TRF0104 TRF0212 Cell Signaling Technology cat. 9224 TRF0219 Carna Biosciences cat. 04-152-10 TRF0213 Carna Biosciences cat. 07-141-10 RRPSYRK VTPRTPPP PRTSSFAE RRRSLLE RKGSLRQ RHDSGLDSM ARTKQTARKSTG TRTDSYSAG FLGFTYVAP RSAMSGLHL YRSPSMPEN (R/K)XXSXP STTPGGTLFSTTPG RRL(pS)SSLRA DEKTDDE www.perkinelmer.com/kinaseconnection LANCE Ultra Tyrosine Kinase Selection Guide How to use this guide: 1.Locate your kinase • In many cases, up to three commercial kinase vendors have been tested. • Many common aliases are shown in parenthesis. 2.Best performing ULight ™ substrates are listed for each enzyme according to performance criteria where signal to background (S/B) ratios are greater than 3.0 • S/B ratios (Signal at 665 nm / minus ATP control at 665 nm) are indicated in parenthesis. • All S/B ratios were obtained at fixed experimental conditions unless otherwise noted (see page 16). If you cannot find your kinase of interest, please ask your PerkinElmer sales specialist, as our list continues to expand. Two kits are available for testing purposes: • KinaSelect Ser/Thr kit (5 x 250 data points, TRF0300-C) – 5 ULight-labeled SER/THR kinase specific substrates + 5 matching Europium-labeled Antiphospho-Antibodies • KinaSelect TK kit (1,000 data points, TRF0301-D) – 1 ULight-labeled kinase specific substrate + 1 matching Europium-labeled Anti-phospho-Antibody 3.Based on your substrate choice, find the corresponding Europium-labeled anti-phospho-antibody on page 16 (LANCE Ultra products used for this study) to identify the TR-FRET donor anti-phospho-antibody for your LANCE Ultra assay. 12 www.perkinelmer.com/kinaseconnection Kinase Kinase Concentration (nM) ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 ABL 0.25 ULight-poly GT (13.1) ULight-poly GAT (11.1) ULight-TK (4.1) ABL [T315I] (ABL1) 0.5 ULight-poly GT (6.6) ULight-poly GAT (6.4) ULight-TK (4.8) ACK 20 ULight-poly GT (27.3) ULight-poly GAT (27.2) ULight-TK (17.3) ALK 4 ULight-IRS-1 (16.8) ULight-poly GT (9.8) ULight-TK (8.8) ARG 0.5 ULight-poly GT (13.2) ULight-poly GAT (12.7) ULight-TK (10.6) AXL 4 ULight-poly GT (9.8) ULight-poly GAT (5.6) BLK 0.5 ULight-TK (31.0) ULight-poly GT (16.5) ULight-CDK1 (9.6) BMX 0.5 ULight-poly GT (13.2) ULight-poly GAT (9.0) ULight-TK (7.1) BRK 3 ULight-poly GT (17.4) ULight-poly GAT (3.0) BTK 0.25 ULight-poly GAT (19.6) ULight-poly GT (11.8) ULight-TK (6.8) ULight-poly GT (10.4) CSK 20 ULight-TK (27.2) ULight-poly GAT (20.3) CTK 20 ULight-poly GAT (18.8) ULight-poly GT (12.6) DDR1 0.5 ULight-poly GAT (3.0) DDR2 0.5 ULight-poly GAT (5.1) ULight-IRS-1 (4.9) ULight-poly GT (4.1) EGFR 0.5 ULight-JAK-1 (19.0) ULight-TK (10.0) ULight-poly GAT (5.0) EGFR [T790M] 0.5 ULight-poly GT (18.1) ULight-poly GAT (10.8) ULight-JAK-1 (3.9) EphA1 0.5 ULight-poly GT (24.1) ULight-poly GAT (11.4) ULight-TK (10.8) EphA2 0.25 ULight-TK (26.9) ULight-poly GT (12.3) ULight-poly GAT (7.1) EphA3 0.5 ULight-TK (22.4) ULight-poly GT (17.4) ULight-poly GAT (8.0) EphA4 0.5 ULight-TK (31.5) ULight-poly GT (12.6) ULight-poly GAT (11.3) EphA5 1 ULight-TK (35.5) ULight-poly GAT (21.9) ULight-JAK-1 (21.2) EphA6 0.5 ULight-poly GAT (15.5) ULight-poly GT (14.2) ULight-TK (5.4) EphA7 0.5 ULight-poly GT (22.7) ULight-TK (17.2) ULight-poly GAT (11.1) EphA8 0.25 ULight-TK (32.3) ULight-poly GT (16.4) ULight-poly GAT (7.8) EphB1 0.5 ULight-TK (29.9) ULight-poly GT (25.1) ULight-CDK1 (14.1) EphB2 0.25 ULight-TK (28.8) ULight-poly GT (27.9) ULight-poly GAT (13.2) EphB3 0.1 ULight-poly GT (29.0) ULight-TK (24.7) ULight-poly GAT (11.2) EphB4 0.5 ULight-TK (29.8) ULight-poly GT (13.7) ULight-CDK1 (12.6) ULight-poly GT (20.0) ULight-poly GAT (13.0) ULight-TK (31) ULight-poly GAT (17.1) FAK 3 FER 0.25 FES ULight-TK (29.4) ULight-poly GAT (19.0) ULight-poly GT (17.1) FGFR1 0.5 ULight-JAK-1 (30.8) ULight-TK (26.4) ULight-IRS-1 (13.5) FGFR2 0.25 ULight-JAK-1 (33.2) ULight-TK (27.1) ULight-IRS-1 (15.5) FGFR3 0.25 ULight-JAK-1 (27.6) ULight-TK (24.4) ULight-poly GT (16.0) FGFR4 3 ULight-JAK-1 (20.8) ULight-TK (17.7) ULight-poly GT (9.2) 13 1 ULight-CDK1 (16.1) www.perkinelmer.com/kinaseconnection Kinase Kinase Concentration (nM) ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 FGR 0.1 ULight-TK (29.3) ULight-poly GT (25.1) ULight-poly GAT (17.7) FLT1 0.25 ULight-poly GT (22.2) ULight-poly GAT (15.4) ULight-JAK-1 (10.0) FLT3 0.1 ULight-TK (25.9) ULight-JAK-1 (25.0) ULight-poly GT (20.8) FLT4 0.25 ULight-JAK-1 (36.9) ULight-TK (20.0) ULight-IRS-1 (11.4) FMS (CSFR) 0.25 ULight-poly GT (14.6) ULight-JAK-1 (6.3) ULight-poly GAT (6.2) FRK 0.5 ULight-poly GAT (22.9) ULight-poly GT (20.8) ULight-TK (9.7) FYN 0.25 ULight-TK (25.9) ULight-CDK1 (19.7) ULight-poly GT (12.8) HCK 0.25 ULight-TK (29.7) ULight-poly GT (21.3) ULight-poly GAT (13.2) HER2 10 ULight-poly GT (9.3) ULight-poly GAT (8.4) HER4 (ERBB4) 0.1 ULight-poly GT (25.2) ULight-poly GAT (14.4) IGF1R 0.25 ULight-TK (25.1) ULight-poly GT (21.8) ULight-IRS-1 (15.2) INSR 20 ULight-poly GAT (25.4) ULight-poly GT (18.3) ULight-TK (4.0) ULight-TK (3.1) IRR 0.5 ULight-poly GT (21.6) ULight-TK (19.2) ULight-IRS-1 (15.6) ITK 0.5 ULight-poly GT (9.8) ULight-poly GAT (8.5) ULight-TK (8.4) JAK1 7 ULight-JAK-1 (19.5) ULight-poly GT (17.6) ULight-poly GAT (16.4) JAK2 1 ULight-JAK-1 (41.8) ULight-TK (35.5) ULight-IRS-1 (21.4) JAK3 0.5 ULight-JAK-1 (37.3) ULight-TK (16.1) ULight-poly GAT (6.9) KDR 0.25 ULight-JAK-1 (30.8) ULight-TK (16.9) ULight-poly GT (8.6) KIT 2 ULight-poly GAT (21.7) ULight-poly GT (13.0) ULight-JAK-1 (3.3) LCK 0.5 ULight-poly GAT (16.5) ULight-TK (12.3) ULight-poly GT (10.4) 1 ULight-poly GAT (25.1) ULight-poly GT (21.3) ULight-TK (13.7) ULight-TK (32.5) ULight-poly GT (15.6) ULight-CDK1 (14.3) ULight-poly GT (18.6) LTK LYNa 0.5 LYNb 1 ULight-TK (33.1) ULight-CDK1 (24.0) MATK (Hyl) 1 ULight-poly GT (20.7) ULight-poly GAT (8.8) MER 0.25 ULight-poly GT (29.2) ULight-poly GAT (15.9) ULight-TK (12.9) MET 0.5 ULight-TK (24.2) ULight-JAK-1 (19.7) ULight-poly GT (17.0) 1 ULight-poly GAT (10.6) ULight-JAK-1 (3.9) ULight-TK (3.1) 0.5 ULight-poly GT (17.8) ULight-poly GAT (10.6) ULight-JAK-1 (9.0) ULight-TK (4.7) MUSK PDGFRa PDGFRb 0.25 ULight-poly GAT (12.5) ULight-poly GT (11.2) PTK2B (FAK2) 0.25 ULight-polyGT (19.2) ULight-poly GAT (12.3) PYK2 0.25 ULight-poly GT (15.5) ULight-poly GAT (7.7) RET 0.5 ULight-JAK-1 (23.6) ULight-TK (20.3) ULight-IRS-1 (6.9) RON 0.25 ULight-poly GT (27.7) ULight-poly GAT (25.7) ULight-JAK-1 (4.9) ROR1 20 ULight-poly GT (3.4) ROR2 20 ULight-poly GT (4.2) Newly added Kinases highlighted in Green 14 www.perkinelmer.com/kinaseconnection Kinase Kinase Concentration (nM) ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 ROS 0.25 ULight-TK (24.5) ULight-poly GT (23.8) ULight-IRS-1 (20.5) SRC 0.5 ULight-TK (31.7) ULight-poly GAT (18.4) ULight-CDK1 (16.6) SRM 0.5 ULight-TK (32.5) ULight-poly GAT (20.1) ULight-poly GT (18.3) SYK 0.5 ULight-TK (24.6) ULight-JAK-1 (20.0) ULight-poly GAT (4.1) TEC 0.5 ULight-poly GT (25.4) ULight-TK (18.6) ULight-poly GAT (15.4) TIE2 0.5 ULight-poly GT (19.3) ULight-TK (17.7) ULight-poly GAT (9.4) TNK1 ULight-poly GAT (15.2) ULight-poly GT (9.3) ULight-TK (6.8) TRKA (NTRK1) 0.5 1 ULight-TK (23.2) ULight-poly GAT (17.5) ULight-poly GT (16.3) TRKB (NTRK2) 0.5 ULight-TK (28.0) ULight-poly GAT (26.3) ULight-poly GT (22.9) TRKC (NTRK3) 0.5 ULight-poly GT (34.1) ULight-poly GAT (22.3) ULight-TK (6.4) TXK 3 ULight-poly GAT (18.9) ULight-poly GT (11.0) ULight-TK (5.9) TYK2 4 ULight-JAK-1 (15.2) ULight-IRS-1 (7.3) ULight-TK (7.3) TYRO3 0.25 ULight-poly GT (21.5) ULight-poly GAT (9.1) ULight-TK (6.1) YES 0.25 ULight-TK (28.0) ULight-poly GT (15.3) ULight-poly GAT (14.0) ULight-poly GAT (15.3) ULight-poly GT (11.1) ULight-TK (7.6) ZAP70 15 4 www.perkinelmer.com/kinaseconnection Kinase assay components: Kinases: see table for concentration ATP: 200 µM in kinase reaction ULight-poly GT, ULight-poly GAT and ULight-IRS-1:100 nM in kinase reaction ULight-JAK-1, ULight-TK and ULight-CDK1: 50 nM in kinase reaction Europium anti-phospho-substrate antibodies: 2 nM in final volume Kinase buffer: 50 mM HEPES pH 7.5 10 mM MgCl2 1 mM EGTA 2 mM DTT and 0.01% Tween 20 Kinase reactions were performed in 10 µL in white OptiPlates™-384 (cat# 6007290). After 2 h incubation at 23 °C, kinase reactions were stopped with EDTA (10 mM final concentration in 20 µL). Eu-anti-phospho antibodies were added and incubated for one hour prior to detection with the EnVision®. All kinases used in the tyrosine study were from commercially available lots purchased from Carna Biosciences. NOTE: Signal to background (S/B) ratios have also been measured in an alternative manner: the control reaction contained ATP, but no substrate, and the substrate was added after stopping the reaction with EDTA. In most cases the S/B ratios are similar using either method. The data with the alternative S/B ratios is available upon request. LANCE Ultra products used for this study: Catalog number Phospho-Motif ULight-poly GT TRF0100 [EY(4:1)]n ULight-poly GAT TRF0101 [EAY(1:1:1)]n ULight-IRS-1 (Tyr983) Peptide TRF0120 SRGDYMTMQ ULight-JAK-1 (Tyr1023) Peptide TRF0121 DKEYYTVKD ULight-CDK1 (Tyr15) Peptide TRF0122 GEGTYVVY ULight-TK Peptide TRF0127 Y Europium-anti-phosphotyrosine (PT66) AD0068, AD0069 Y KinaSelect TK kit* TRF0301-D Y *contains ULight-TK Peptide, 0.5 nmole, Europium-anti-phosphotyrosine (PT66), 10 µg and Lance Detection Buffer 10X, 1.8 mL PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2010, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 008999_01