FINDING THE PATHWAY TO ASSAY OPTIMIZATION IS EASY LANCE® Ultra 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: – 225 Serine/Threonine kinases validated on LANCE Ultra reagents – 85 Tyrosine kinases validated on LANCE Ultra reagents 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 • Signal to background (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 anti-phospho 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 (i.e. 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/lance 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 ULight Substrate 2 ULight Substrate 3 ACTR2B (ACVR2B)ULight-Histone H3 (Thr3; 10.4) ULight-Topo IIa (7.2) ACV-R1 (ALK2)ULight-Topo IIa (10.9) ALK2 (ACV-R1)ULight-Topo IIa (Mn2+; 21.5) ACVR2AULight-Histone H3 (Thr3; 9.4) ULight-CREBtide (5.7) ACVRL1 (ALK1)ULight-CREBtide (14.2) ULight-Topo IIa (5.6) ULight-PLK (5.4) BARK1 (ADRBK1, GRK2)ULight-Topo IIa (22.9) ADRBK2 (BARK2, GRK3)ULight-Topo IIa (27.9) BARK2 (ADRBK2, GRK3)ULight-Topo IIa (22.9) ULight-Histone H3 (Thr3; 10.1) AKT1ULight-CREBtide (32.0) ULight-rpS6 (20.5) ULight-PLK (19.1) AKT2ULight-CREBtide (32.4) ULight-rpS6 (21.7) ULight-PLK (20.1) AKT3ULight-CREBtide (32.1) ULight-rpS6 (21.6) ULight-PLK (20.7) ALK4 (ACVR1B)ULight-Topo IIa (20.6) AMPK-alpha1 flULight-CREBtide (9.6) ULight-Histone H3 (Thr3; 5.3) AMPKa1/b1/g1 (PRKAA1/B1/G1)ULight-CREBtide (31.9) ULight-Acetyl-CoA Carboxylase (30.5) ULight-PLK (15.1) AMPKa2/b1/g1(PRKAA2/B1/G1) ULight-CREBtide (30.3) ULight-Acetyl-CoA Carboxylase (29.9) ULight-PLK (11.5) ARK5 (NuaK1)ULight-CREBtide (18.8) ULight-Topo IIa (7.7) NuaK1 (ARK5)ULight-CREBtide (22.8) ULight-Topo IIa (3.1) AurA (Aurora-A)ULight-PLK (20.5) ULight-Histone H3 (Ser10; 13.0) AurB (AURKB)/INCENPULight-CREBtide (29.8) ULight-rpS6 (21.0) ULight-PLK (17.6) Aurora-BULight-CREBtide (13.4) ULight-Histone H3 (Ser10; 12.7) ULight-PLK (7.6) AurC (AURKC)ULight-PLK (16.2) ULight-Histone H3 (Ser10; 12.1) ULight-CREBtide (11.7) Aurora-CULight-CREBtide (8.9) ULight-Histone H3 (Ser10; 8.4) ULight-PLK (3.8) BMPR1BULight-Topo IIa (18.7) BMPR2ULight-Histone H3 (Thr3; 11.0) BRAF GST-MAP2K1/ ULight anti-GST (6.1) BRAF [V600E] GST-MAP2K1/ ULight anti-GST (10.5) BRSK1ULight-CDC25 (7.6) ULight-Acetyl-CoA Carboxylase (5.2) BRSK2ULight-CDC25 (8.4) ULight-Acetyl-CoA Carboxylase (7.9) CaMK1a (CAMK1)2ULight-CREBtide (28.4) CaMK1b (PNCK)2ULight-CREBtide (29.6) CaMK1d (CAMK1D)2ULight-CREBtide (30.5) CaMK2a (CAMK2A)2ULight-CREBtide (26.1) ULight-rpS6 (22.3) ULight-PKC (16.1) CaMK2b (CAMK2B)2ULight-CREBtide (30.7) ULight-rpS6 (23.6) ULight-mTOR (12.0) CaMK2d (CAMK2D)2ULight-rpS6 (25.4) ULight-CREBtide (23.8) ULight-PLK (14.0) CaMK2g (CAMK2G)2ULight-CREBtide (26.2) ULight-rpS6 (18.2) ULight-PKC (15.6) Kinase Supplier Supplier Cat. No. Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Invitrogen Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 09-132 0372-0000-1 09-134 09-131 PV4883 01-111 PV3827 01-112 01-101 01-102 01-103 09-135 0289-0000-1 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 09-122 09-144 02-115 02-116 02-104 Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 02-105 02-106 02-109 02-110 02-111 02-112 Newly added Kinases highlighted in Green 3 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 CaMK4 ULight-CREBtide (33.5) ULight-mTOR (8.6) ULight-PLK (6.0) CAMKK12ULight-p70S6K (25.1) ULight-CREBtide (19.6) CAMKK2ULight-Histone H3 (Thr3; 12.2) CAMKK22ULight-Histone H3 (Thr3; 12.0) ULight-p70S6K (5.5) CDC2/CycB1ULight-MBP (43.2) ULight-4E-BP1 (37.2) CDC2L6/CycCULight-Histone H3 (Thr3; 10.0) CDC42BPB (MRCKb)ULight-Histone H3 (Ser10; 6.7) MRCKb (CDC42BPB)ULight-Histone H3 (Ser10; 6.9) ULight-CREBtide (6.3) ULight-PLK (3.3) CDC7/ASKULight-Topo IIa (14.6) CDK1/CycAULight-4E-BP1 (37.9) ULight-MBP (21.9) CDK1/CycB1ULight-4E-BP1 (39.6) ULight-MBP (27.8) CDK1/CycEULight-4E-BP1 (39.2) ULight-MBP (21.5) CDK2/CycAULight-MBP (39.9) ULight-4E-BP1 (36.7) CDK2/CycE1ULight-4E-BP1 (33.5) ULight-MBP (28.5) CDK3/CycE1ULight-MBP (38.9) ULight-4E-BP1 (23.3) CDK4/CycD1ULight-4E-BP1 (38.7) ULight-MBP (21.6) CDK4/CycD3ULight-MBP (37.2) ULight-4E-BP1 (32.3) CDK5/p25ULight-MBP (37.1) ULight-4E-BP1 (36.5) CDK5/p25NCKULight-4E-BP1 (34.2) ULight-MBP (24.9) CDK5/p35NCKULight-4E-BP1 (40.3) ULight-MBP (22.9) CDK6/CycD1ULight-4E-BP1 (39.6) ULight-MBP (17.0) CDK6/CycD3ULight-MBP (35.7) ULight-4E-BP1 (35.4) CDK7/CycH/MAT1ULight-MBP (19.0) CDK8/CycC ULight-CREBtide (7.3) ULight-GS (5.0) ULight-rpS6 (4.7) CDK9/CyclinK ULight-GS (9.3) CDK9/CycT1ULight-4E-BP1 (28.1) ULight-MBP (14.4) CGK2 (PKG2, PRKG2)ULight-CREBtide (35.8) ULight-PLK (14.1) CHK1 (CHEK1)ULight-CREBtide (30.4) ULight-PKC (15.7) ULight-Acetyl-CoA Carboxylase (8.1) CHK2 (CHEK2)ULight-CREBtide (31.8) ULight-PLK (7.3) ULight-rpS6 (6.4) CK1a (CSNK1A1)ULight-Topo IIa (13.7) CK1-alpha1ULight-Topo IIa (15.0) CK1d (CSNK1D)ULight-Topo IIa (23.5) ULight-GS (12.5) CK1e (CSNK1E)ULight-Topo IIa (23.7) CK1g1 (CSNK1G1)ULight-Histone H3 (Thr3; 14.6) CK1g2 (CSNK1G2)ULight-Histone H3 (Thr3; 10.1) ULight-Topo IIa (7.2) CK1g3 (CSNK1G3)ULight-Histone H3 (Thr3; 12.9) CK2-alpha1ULight-Topo IIa (22.0) CK2a1/b (CSNK2A1/B)ULight-IkBa (28.7) ULight-Topo IIa (13.8) ULight-Histone H3 (Thr3; 10.9) CK2-alpha2ULight-Topo IIa (21.4) CK2a2/b (CSNK2A2/B)ULight-IkBa (29.6) ULight-Topo IIa (14.6) ULight-Histone H3 (Thr3; 10.1) CLK1ULight-MBP (41.2) ULight-4E-BP1 (25.7) ULight-GS (20.1) CLK2ULight-CREBtide (19.1) ULight-MBP (11.9) ULight-Histone H3 (Ser10; 8.3) 2 Kinase Supplier Supplier Cat. No. Carna Biosciences 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 ProQinase Invitrogen Carna Biosciences Carna Biosciences 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 Carna Biosciences 02-108 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 0376-0390-1 PV4335 04-110 01-143 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 04-127 Newly added Kinases highlighted in Green 4 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 CLK3ULight-CREBtide (26.2) ULight-GS (14.1) ULight-rpS6 (8.5) COT (MAP3K8)ULight-p70S6K (Mn2+; 11.0) ULight-Acetyl-CoA Carboxylase (Mn2+; 7.8) GST-MAP2K1 / ULight-anti-GST (Mn2+; 7.8) COT GST-MAP2K1 / ULight-anti-GST (Mn2+; 8.0) ULight-p70S6K (Mn2+; 5.3) ULight-Acetyl-CoA Carboxylase (Mn2+; 4.3) CRIK (CIT)ULight-Histone H3 (Ser10; 8.3) DAPK1ULight-CREBtide (23.7) ULight-PLK (18.1) ULight-Histone H3 (Ser10; 9.3) DAPK1ULight-CREBtide (10.5) ULight-Histone H3 (Thr3; 9.8) DAPK2ULight-CREBtide (6.7) ULight-PLK (6.4) DAPK3 ULight-CREBtide (13.8) ULight-Histone H3 (Thr3; 6.6) ULight-Histone H3 (Ser10; 6.3) DCAMKL1ULight-CREBtide (34.8) DCAMKL2ULight-CREBtide (31.4) DLK (MAP3K12)ULight-CREBtide (36.4) ULight-PKC (11.6) ULight-Histone H3 (Thr3; 10.9) DNA PK3ULight-mTOR (40 U; S/B +/- DNA: 6.6) DRAK1 (STK17A)ULight-CREBtide (9.2) ULight-Histone H3 (Thr3; 6.2) DYRK1AULight-GS (20.1) ULight-MBP (18.5) ULight-4E-BP1 (7.4) DYRK1BULight-4E-BP1 (24.6) ULight-MBP (23.2) ULight-GS (19.2) DYRK2ULight-MBP (33.4) ULight-4E-BP1 (30.0) ULight-GS (19.9) DYRK3ULight-4E-BP1 (39.9) ULight-MBP (22.0) ULight-GS (18.3) DYRK4ULight-4E-BP1 (36.3) ULight-MBP (26.7) ULight-GS (20.2) Erk1 (MAPK3)ULight-4E-BP1 (37.9) GST-ATF-2 / ULight-anti-GST (37.6) ULight-MBP (26.6) Erk2 (MAPK1)ULight-4E-BP1 (37.1) GST-ATF-2 / ULight-anti-GST (35.1) ULight-MBP (28.0) ERK2 GST-ATF-2 / ULight-anti-GST (40.0) ULight-4E-BP1 (31.9) ULight-MBP (19.4) Erk5 (MAPK7)ULight-4E-BP1 (22.4) Erk7 (MAPK15)ULight-4E-BP1 (22.3) GCN2ULight-CREBtide (13.5) ULight-Histone H3 (Thr3; 12.3) GPRK4 (GRK4)ULight-Topo IIa (18.8) ULight-CREBtide (15.7) ULight-p70S6K (14.2) GPRK6 (GRK6)ULight-Histone H3 (Thr3; 10.8) ULight-Topo IIa (4.3) GPRK7 (GRK7)ULight-Histone H3 (Thr3; 10.2) ULight-Topo IIa (5.9) GRK5ULight-IkBa (26.5) ULight-Topo IIa (26.3) ULight-Histone H3 (Thr3; 11.7) GSK3a (GSK3A)ULight-4E-BP1 (34.5) ULight-GS (21.5) GSK3b (GSK3B)ULight-4E-BP1 (35.9) ULight-GS (21.9) Haspin (GSG2)ULight-Histone H3 (Thr3; 15.5) HGK (MAP4K4)ULight-p70S6K (23.8) HIPK1ULight-4E-BP1 (30.8) ULight-MBP (22.9) ULight-GS (20.1) HIPK2ULight-MBP (37.4) ULight-GS (17.4) HIPK3ULight-4E-BP1 (36.9) ULight-MBP (22.9) ULight-GS (21.3) HIPK4ULight-4E-BP1 (37.7) ULight-MBP (21.2) ULight-GS (20.5) HRIULight-CREBtide (13.3) ULight-PLK (6.2) ICKULight-MBP (33.4) ULight-4E-BP1 (24.7) ULight-GS (12.6) IKKa (CHUK)ULight-IkBa (22.4) ULight-rpS6 (17.9) ULight-Acetyl-CoA Carboxylase (11.8) IKKbULight-p70S6K (30.4) ULight-IkBa (25.6) ULight-rpS6 (22.1) IKK-betaULight-IkBa (25.5) IKKe (IKBKE)ULight-p70S6K (22.1) ULight-rpS6 (20.2) ULight-PLK (15.8) Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences ProQinase 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 Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences 04-128 07-301 0180-0000-1 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 05-153 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 04-156 05-112 05-084 0258-0000-1 05-114 Newly added Kinases highlighted in Green 5 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 IKK-epsilonULight-rpS6 (22.0) ULight-p70S6K (15.5) ULight-PLK (12.5) IRAK1ULight-p70S6K (28.3) ULight-Acetyl-CoA Carboxylase (17.7) ULight-Histone H3 (Thr3; 14.4) IRAK4ULight-p70S6K (Mn2+; 32.4) ULight-Acetyl-CoA Carboxylase (31.9) IRAK4ULight-p70S6K (Mn2+; 28.1) ULight-Acetyl-CoA Carboxylase (23.9) JNK1 (MAPK8)ULight-4E-BP1 (34.5) GST-ATF-2 / ULight-anti-GST (32.0) ULight-MBP (21.4) JNK2 (MAPK9)ULight-4E-BP1 (35.0) GST-ATF-2 / ULight-anti-GST (32.8) ULight-MBP (20.8) JNK3 (MAPK10) GST-ATF-2 / ULight-anti-GST (33.7) ULight-4E-BP1 (32.8) ULight-MBP (19.8) LATS2ULight-CREBtide (14.8) ULight-mTOR (5.0) LRRK2ULight-CREBtide (24.2) ULight-PLK (19.9) ULight-Histone H3 (Thr3; 10.4) LRRK2 [G2019S]ULight-Histone H3 (Thr3; 15.4) LRRK2 [G2019S]ULight-Histone H3 (Thr3; 15.3) ULight-CREBtide (8.4) LZKULight-CREBtide (17.9) ULight-Histone H3 (Thr3; 8.3) MAP2K3GST-p38a / ULight-anti-GST (19.8) MAP2K4GST-p38a / ULight-anti-GST (22.1) MAP2K6 (MKK6)GST-p38a/ ULight-anti-GST (23.1) MAP3K1ULight-Acetyl-CoA Carboxylase (5.3) MAP3K2 GST-MAP2K1 / ULight-anti-GST (5.9) MAP3K6ULight-CREBtide (6.5) MAP3K14 (NIK)ULight-CREBtide (9.3) NIK (MAP3K14)ULight-CREBtide (8.5) MAP4K2ULight-rpS6 (18.3) ULight-CREBtide (16.0) ULight-p70S6K (9.2) MAP4K5 (KHS1)ULight-p70S6K (35.6) ULight-rpS6 (24.9) ULight-Acetyl-CoA Carboxylase (23.8) MAPKAPK2ULight-4E-BP1 (32.2) ULight-MBP (31.4) ULight-CREBtide (29.1) MAPKAPK3ULight-CREBtide (27.1) ULight-rpS6 (13.2) ULight-PKC (12.3) MAPKAPK3ULight-CREBtide (26.1) ULight-mTOR (11.8) ULight-CDC25 (8.2) MAPKAPK5ULight-MBP (11.1) ULight-4E-BP1 (10.1) MAPKAPK5ULight-Topo IIa (3.6) ULight-PLK (3.6) MARK1ULight-PLK (18.1) ULight-Acetyl-CoA Carboxylase (13.9) ULight-CREBtide (8.9) MARK1ULight-Acetyl-CoA Carboxylase (9.4) ULight-PLK (9.3) ULight-CREBtide (8.0) MARK2 (EMK1)ULight-PLK (18.0) ULight-Acetyl-CoA Carboxylase (15.6) ULight-CREBtide (8.5) MARK3 (C-TAK1)ULight-PLK (12.3) ULight-Acetyl-CoA Carboxylase (11.9) ULight-CDC25 (7.6) MARK3 (C-TAK1)ULight-Acetyl-CoA Carboxylase (15.1) ULight-PLK (13.2) ULight-CDC25 (7.1) MARK4 (MARKL1)ULight-Acetyl-CoA Carboxylase (25.7) ULight-CREBtide (21.0) ULight-PLK (18.8) MELKULight-CREBtide (32.9) MGC42105 (B3KVM1)ULight-CREBtide (25.8) ULight-PLK (10.8) ULight-CDC25 (7.4) MINK (MINK1)ULight-p70S6K (55.1) ULight-PKC (14.5) ULight-rpS6 (11.8) MLK1 (MAP3K9) GST-MAP2K1 / ULight-anti-GST (11.4) ULight-rpS6 (5.4) MLK2 (MAP3K10)ULight-Histone H3 (Thr3; 11.0) GST-MAP2K1 / ULight-anti-GST (3.2) MLK3 (MAP3K11) GST-MAP2K1 / ULight-anti-GST (6.4) MNK1 (MKNK1)ULight-CREBtide (36.8) ULight-Histone H3 (Ser10; 9.9) MNK2 (MKNK2)ULight-CREBtide (30.5) ULight-rpS6 (18.9) ULight-Histone H3 (Ser10; 13.2) MOSULight-Topo IIa (9.1) ULight-CREBtide (6.6) GST-MAP2K1 / ULight-anti-GST (6.3) Kinase Supplier Supplier Cat. No. ProQinase 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 Carna Biosciences ProQinase Carna Biosciences Invitrogen Carna Biosciences Carna Biosciences ProQinase 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 0320-0000-1 09-101 09-145 0586-0000-1 04-163 04-164 04-150 01-124 09-110 PV4881 09-146 09-114 07-048 07-044 07-046 07-103 07-004 07-108 07-102 0344-0000-1 07-111 PV3682 02-142 02-143 0633-0000-1 02-144 0380-0000-1 02-120 0432-0000-1 02-121 02-122 0433-0000-1 02-123 02-124 02-125 07-139 09-015 09-116 09-017 02-145 02-146 05-118 Newly added Kinases highlighted in Green 6 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 MRCKa (CDC42BPA)ULight-Histone H3 (Ser10; 7.5) ULight-CREBtide (6.7) MSK1 (RPS6KA5)ULight-CREBtide (28.5) ULight-rpS6 (26.4) ULight-PLK (20.6) MSK2 (RPS6KA4)ULight-CREBtide (31.1) ULight-rpS6 (25.0) ULight-PLK (19.8) MST1 (STK4)ULight-p70S6K (13.3) ULight-PKC (12.8) ULight-Histone H3 (Thr3; 8.9) MST1 (STK4)ULight-PKC (11.9) ULight-Histone H3 (Thr3; 8.4) ULight-p70S6K (6.5) MST1 (STK4)ULight-PKC (14.0) ULight-Histone H3 (Thr3; 6.5) ULight-rpS6 (5.5) MST2 (STK3)ULight-Histone H3 (Thr3; 8.0) ULight-PKC (5.2) ULight-p70S6K (4.4) MST2 (STK3)ULight-p70S6K (12.5) ULight-PKC (9.0) ULight-Histone H3 (Thr3; 6.2) MST3 (STK24)ULight-CREBtide (14.7) ULight-PKC (9.3) ULight-rpS6 (8.9) MST4 (MASK)ULight-PKC (14.6) ULight-rpS6 (8.5) mTOR (1362-end)ULight-4E-BP1 (Mn2+; 36.9) ULight-p70S6K (Mn2+; 18.3) MYT1 (PKMYT1)ULight-CREBtide (10.6) NDR1 (STK38)ULight-CREBtide (10.5) ULight-Histone H3 (Ser10; 8.7) NDR2ULight-Histone H3 (Ser10; 8.0) ULight-CREBtide (7.0) NEK1ULight-p70S6K (60.0) ULight-Acetyl-CoA Carboxylase (24.0) ULight-rpS6 (18.6) NEK2ULight-p70S6K (43.3) ULight-CREBtide (31.2) ULight-rpS6 (18.2) NEK3ULight-p70S6K (26.9) ULight-4E-BP1 (18.2) NEK4ULight-p70S6K (40.1) NEK6ULight-p70S6K (32.0) NEK7ULight-p70S6K (33.4) NEK9ULight-p70S6K (48.9) ULight-rpS6 (16.5) ULight-Acetyl-CoA Carboxylase (13.0) NEK11ULight-p70S6K (20.0) ULight-Histone H3 (Thr3; 14.9) NLKULight-CREBtide (9.4) ULight-PLK (1.3) NLKULight-CREBtide (15.4) ULight-PLK (6.0) Nuak2 (SNARK)ULight-CREBtide (23.9) ULight-Histone H3 (Thr3; 7.2) SNARK (Nuak2)ULight-CREBtide (14.8) ULight-Histone H3 (Thr3; 5.8) ULight-PLK (5.5) OSR1 (OXSR1)ULight-Histone H3 (Thr3; 5.6) p38a (MAPK14)ULight-4E-BP1 (37.0) GST-ATF-2 / ULight-anti-GST (32.1) ULight-MBP (27.9) p38-alpha (MAPK14) GST-ATF-2 / ULight-anti-GST (36.7) ULight-4E-BP1 (33.1) ULight-MBP (5.4) p38b (MAPK11)ULight-MBP (40.6) GST-ATF-2 / ULight-anti-GST (36.9) ULight-4E-BP1 (32.6) p38-beta (MAPK11) GST-ATF-2 / ULight-anti-GST (36.8) ULight-4E-BP1 (35.8) ULight-MBP (14.2) p38d (MAPK13)ULight-MBP (41.2) GST-ATF-2 / ULight-anti-GST (35.1) ULight-4E-BP1 (30.7) p38g (MAPK12)ULight-4E-BP1 (37.7) GST-ATF-2 / ULight-anti-GST (37.0) ULight-MBP (33.6) p70S6K (S6K; RPS6KB1)ULight-CREBtide (30.2) ULight-rpS6 (18.1) ULight-Histone H3 (Ser10; 9.4) p70S6Kb (RPS6KB2)ULight-CREBtide (28.6) ULight-rpS6 (23.0) ULight-Crosstide (13.9) PAK1ULight-PLK (12.3) ULight-Histone H3 (Ser10; 4.5) PAK1ULight-PLK (15.6) PAK2ULight-PLK (16.7) ULight-Histone H3 (Ser10; 4.9) PAK3ULight-PLK (19.9) ULight-Histone H3 (Ser10; 12.2) ULight-GS (9.2) PAK4ULight-CREBtide (12.2) ULight-PLK (4.3) PAK5 (PAK7)ULight-PLK (18.5) PAK61ULight-PLK (5h; 7.0) Kinase Supplier Supplier Cat. No. Carna Biosciences Carna Biosciences Carna Biosciences Millipore Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Millipore Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 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 Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences 01-107 01-147 01-148 14-624 07-116 0667-0000-1 07-117 0699-0000-1 07-118 07-119 14-770 05-176 01-125 01-126 05-123 05-226 05-127 05-128 05-130 05-131 05-133 05-125 04-151 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 01-155 07-123 0357-0000-1 07-124 07-125 0365-0000-1 07-127 07-128 Newly added Kinases highlighted in Green 7 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 PAK6ULight-Histone H3 (Thr3; 8.5) ULight-PLK (5.5) PASKULight-CREBtide (31.0) ULight-Crosstide (12.6) ULight-mTOR (10.4) PBKULight-CREBtide (16.3) PBK1ULight-Acetyl-CoA Carboxylase (5 h; 7.1) PEK (EIF2AK3)ULight-p70S6K (51.9) ULight-Topo IIa (18.0) ULight-Acetyl-CoA Carboxylase (15.4) PGK (PRKG1)ULight-CREBtide (37.0) ULight-PLK (11.1) PHKG1ULight-CREBtide (31.3) ULight-Histone H3 (Thr3; 8.5) ULight-rpS6 (7.0) PHKG2ULight-CREBtide (31.7) ULight-rpS6 (15.6) ULight-mTOR (9.7) PIM1ULight-CREBtide (34.7) ULight-mTOR (8.5) ULight-rpS6 (8.2) PIM1ULight-CREBtide (31.2) ULight-rpS6 (21.5) ULight-mTOR (12.6) PIM2ULight-CREBtide (30.3) ULight-rpS6 (20.9) ULight-mTOR (11.3) PIM2ULight-CREBtide (14.6) ULight-rpS6 (10.4) ULight-mTOR (6.6) PIM3ULight-CREBtide (31.0) ULight-rpS6 (24.7) ULight-mTOR (11.7) PKACa (PRKACA)ULight-CREBtide (24.3) ULight-rpS6 (16.8) ULight-PLK (16.6) PKACb (PRKACB)ULight-rpS6 (23.7) ULight-PLK (20.8) ULight-CREBtide (15.7) PKCa (PRKCA)ULight-CREBtide (28.1) ULight-rpS6 (19.4) ULight-PKC (14.5) PKCb1 (PRKCB1)ULight-CREBtide (30.5) ULight-PKC (15.6) PKCb2 (PRKCB2)ULight-CREBtide (26.1) ULight-rpS6 (19.1) ULight-PKC (14.0) PKC-beta2 (PRKCB2)ULight-CREBtide (31.4) ULight-PKC (16.2) ULight-rpS6 (2.9) PKCd (PRKCD)ULight-CREBtide (29.4) ULight-PKC (14.8) PKCe (PRKCE)ULight-CREBtide (31.0) ULight-PKC (14.9) PKACgULight-CREBtide (33.4) ULight-rpS6 (24.2) ULight-PLK (21.3) PKCg (PRKCG)ULight-CREBtide (29.7) ULight-PKC (15.6) ULight-rpS6 (9.1) PKC-gamma (PRKCG)ULight-CREBtide (26.1) ULight-PKC (13.5) ULight-rpS6 (10.2) PKCh (PRKCH)ULight-CREBtide (29.5) ULight-PKC (14.3) ULight-rpS6 (9.5) PKC-eta (PRKCH)ULight-rpS6 (22.4) ULight-PLK (13.2) ULight-PKC (10.6) PKCi (PRKCI)ULight-CREBtide (29.4) ULight-PKC (15.4) ULight-rpS6 (8.9) PKCu (PRKCQ)ULight-CREBtide (30.4) ULight-PKC (15.1) ULight-rpS6 (12.3) PKCz (PRKCZ)ULight-CREBtide (22.4) ULight-PKC (14.1) ULight-rpS6 (12.9) PKC-zetaULight-CREBtide (18.6) ULight-PKC (14.1) PKC-mu (PKD-1, PRKD1)ULight-CREBtide (23.2) ULight-PLK (8.4) ULight-PKC (5.6) PKD1 (PRKD1, PKC-mu)ULight-CREBtide (21.6) ULight-PLK (14.4) PKC-nu (PRKD3, EPK2)ULight-CREBtide (23.1) ULight-PLK (5.4) PKD3 (PRKD3, PKC-nu)ULight-CREBtide (31.4) ULight-Histone H3 (Thr3; 8.8) ULight-PLK (6.0) PKD2 (PRKD2)ULight-PLK (10.3) ULight-CREBtide (9.4) PKN1 (PRK1)ULight-CREBtide (32.6) ULight-PKC (10.5) ULight-rpS6 (7.5) PRK1 (PKN1)ULight-CREBtide (31.8) ULight-rpS6 (17.9) ULight-PKC (14.0) PKN2ULight-CREBtide (32.3) PKN3ULight-CREBtide (33.5) ULight-rpS6 (20.0) ULight-PKC (15.4) PKR (EIF2AK2)ULight-Histone H3 (Thr3; 10.8) ULight-CREBtide (6.7) PLK1ULight-p70S6K (24.8) ULight-Topo IIa (23.0) ULight-Histone H3 (Thr3; 13.4) PLK1ULight-Topo IIa (7.0) ULight-Histone H3 (Thr3; 5.7) Kinase Supplier Supplier Cat. No. ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase ProQinase Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences ProQinase 0423-0000-1 02-128 0185-0000-1 05-168 05-155 01-142 02-152 02-153 02-054 0186-0000-1 02-155 0223-0000-1 02-156 01-127 01-128 01-133 01-134 01-165 0116-0000-1 01-135 01-136 01-129 01-137 0204-0000-1 01-138) 0420-0000-1 01-139 01-140 01-141 0117-0000-1 0115-0000-1 02-157 0661-0000-1 02-159 02-158 01-144 0207-0000-1 01-145 01-146 05-156 05-157 0183-0000-1 Newly added Kinases highlighted in Green 8 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 PLK2 (SNK)ULight-Topo IIa (22.9) ULight-CREBtide (11.6) PLK3ULight-IkBa (27.5) ULight-Topo IIa (20.4) ULight-4E-BP1 (15.0) PLK3ULight-Topo IIa (18.6) ULight-Histone H3 (Thr3; 6.1) ULight-4E-BP1 (5.1) PLK4 (SAK)ULight-CREBtide (10.4) ULight-PLK (1.9) SAK (PLK4) ULight-CREBtide (11.4) ULight-PLK (5.8) PRKXULight-CREBtide (34.1) ULight-PLK (19.7) ULight-PKC (13.7) QIK (SNF1LK2) ULight-CREBtide (29.6) ULight-Acetyl-CoA Carboxylase (28.8) ULight-PLK (14.1) SNF1LK2 (QIK)ULight-CREBtide (32.2) ULight-Acetyl-CoA Carboxylase (24.8) ULight-PLK (14.5) RAF1 GST-MAP2K1/ ULight-anti-GST (9.3) RIPK2ULight-CREBtide (8.4) RIPK5ULight-CREBtide (9.7) ULight-p70S6K (5.7) ROCK1ULight-CREBtide (34.8) ULight-rpS6 (23.4) ULight-PLK (19.6) ROCK2ULight-CREBtide (32.7) ULight-rpS6 (20.0) ULight-PLK (16.9) RSK1 (RPS6KA1)ULight-CREBtide (32.1) ULight-rpS6 (22.0) ULight-PLK (20.5) RSK2 (RPS6KA3)ULight-CREBtide (30.0) ULight-rpS6 (20.9) ULight-Crosstide (18.8) RSK3 (RPS6KA2)ULight-CREBtide (32.0) ULight-rpS6 (25.9) ULight-PLK (18.5) RSK4 (RPS6KA6)ULight-CREBtide (32.0) ULight-rpS6 (23.8) ULight-PLK (18.4) SGKULight-CREBtide (32.7) ULight-rpS6 (20.8) ULight-PLK (20.6) SGK2ULight-CREBtide (32.5) ULight-PLK (21.3) ULight-rpS6 (21.1) SGK3 (SGKL)ULight-CREBtide (28.0) ULight-rpS6 (22.7) ULight-PLK (17.7) SIKULight-CREBtide (17.8) ULight-Acetyl-CoA Carboxylase (9.2) ULight-PLK (7.4) SLKULight-CREBtide (31.0) skMLCK (MYLK)2ULight-CREBtide (35.8) SRPK1ULight-CREBtide (20.0) SRPK2ULight-CREBtide (14.3) STK33ULight-rpS6 (13.1) ULight-mTOR (9.5) ULight-CREBtide (8.0) STK33ULight-CREBtide (8.3) TAK1-TAB1 (MAP3K7)ULight-p70S6K (5.6) TAOK2ULight-p70S6K (15.6) ULight-CREBtide (9.0) ULight-PKC (5.7) TAOK3ULight-CREBtide (21.2) ULight-Histone H3 (Thr3; 9.3) ULight-p70S6K (7.9) TBK1ULight-p70S6K (34.5) ULight-rpS6 (24.8) ULight-Acetyl-CoA Carboxylase (24.5) TGFßR1 (TGFBR1)ULight-Topo IIa (21.3) TGFßR2 (TGFBR2)ULight-CREBtide (6.7) TLK1ULight-Topo IIa (17.4) ULight-Histone H3 (Thr3; 15.2) ULight-p70S6K (9.4) TLK2ULight-Topo IIa (17.9) ULight-Histone H3 (Thr3; 17.2) ULight-p70S6K (13.8) TSK2 (TSSK2)ULight-CREBtide (32.1) ULight-PLK (20.2) ULight-Acetyl-CoA Carboxylase (15.6) TSSK2 (TSK2)ULight-CREBtide (31.0) ULight-Acetyl-CoA Carboxylase (17.9) ULight-PLK (16.2) TSSK1ULight-CREBtide (20.4) ULight-PLK (17.9) ULight-Acetyl-CoA Carboxylase (16.2) TSSK3ULight-CREBtide (32.5) ULight-Acetyl-CoA Carboxylase (27.3) ULight-PLK (20.0) TTBK1ULight-Topo IIa (24.9) ULight-Histone H3 (Thr3; 17.8) ULight-p70S6K (13.4) TTBK2ULight-Topo IIa (24.2) ULight-Histone H3 (Thr3; 17.4) ULight-rpS6 (10.3) ULK3ULight-Histone H3 (Thr3; 6.5) Kinase Supplier Supplier Cat. No. Carna Biosciences 05-158 Carna Biosciences 05-159 ProQinase 0437-0000-1 Carna Biosciences 05-160 ProQinase 0306-0000-1 Carna Biosciences 01-130 Carna Biosciences 02-129 Invitrogen PV4792 Carna Biosciences 09-125 Carna Biosciences 09-128 Invitrogen PV4678 Carna Biosciences 01-109 Carna Biosciences 01-110 Carna Biosciences 01-149 Carna Biosciences 01-150 Carna Biosciences 01-151 Carna Biosciences 01-152 Carna Biosciences 01-158 Carna Biosciences 01-159 Carna Biosciences 01-160 Carna Biosciences 02-131 Carna Biosciences 07-129 Carna Biosciences 02-150 ProQinase 0434-0000-1 ProQinase 0381-0000-1 Millipore14-671 Carna Biosciences 02-301 Carna Biosciences 09-019 Carna Biosciences 07-133 Carna Biosciences 07-135 Carna Biosciences 05-115 Carna Biosciences 09-141 Carna Biosciences 09-142 Carna Biosciences 05-166 Carna Biosciences 05-167 Carna Biosciences 02-165 ProQinase 0220-0000-1 Carna Biosciences 02-364 Carna Biosciences 02-166 Carna Biosciences 03-108 Carna Biosciences 03-109 Carna Biosciences 05-173 Newly added Kinases highlighted in Green 9 www.perkinelmer.com/lance Kinase ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 VRK1ULight-CREBtide (17.0) ULight-Histone H3 (Thr3; 8.7) VRK2ULight-Histone H3 (Thr3; 15.1) WEEULight-CREBtide (10.0) WNK1ULight-CREBtide (7.4) WNK2ULight-CREBtide (21.4) WNK3ULight-CREBtide (4.8) WNK4ULight-CREBtide (10.3) YSK1 (STK25)ULight-CREBtide (21.8) ULight-rpS6 (14.3) ULight-p70S6K (5.7) ZAKULight-p70S6K (36.1) ULight-Histone H3 (Thr3; 15.5) ULight-mTOR (11.4) Kinase Supplier Supplier Cat. No. ProQinase Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences Carna Biosciences 0302-0000-1 03-111 05-177 05-179 05-180 05-181 05-182 07-136 09-120 Kinase reaction time is 5h CaCl2 and Calmodulin at 1 mM and 0.5 µM, 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. 1 2 Kinase assay components: Kinases: 20 nM (various suppliers) ATP: 200 µM ULight substrates in kinase reactions: 50 nM (100 nM for ULight-CDC25) GST-protein substrates in kinase reactions: 30 nM (when indicated) ULight-anti-GST antibody in assay: 30 nM (when indicated) Europium anti-phospho-substrate antibodies in assay: 2 nM Kinase buffer (pH 7.5): 50 mM HEPES 10 mM MgCl2 3 mM MnCl2 (when indicated) 1 mM EGTA 2 mM DTT and 0.01% Tween 20 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. 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 that the assay signal is specific to the enzyme tested. Newly added Kinases highlighted in Green 10 www.perkinelmer.com/lance LANCE® Ultra products used for this study: Labeled Antibody Description ULight-CREBtideULight-CREBtide (Ser133) Europium-anti-phospho-CREB Europium-anti-phospho-CREB (Ser133) ULight-MBPULight-Myelin Basic Protein Peptide Europium-anti-phospho-MBP Europium-anti-phospho-Myelin Basic Protein ULight-CrosstideULight-Crosstide (GSK-3a Ser21) Europium-anti-phospho-Crosstide Europium-anti-phospho-Crosstide (GSK-3a Ser21) ULight-PLKULight-PLK (Ser137) Peptide Europium-anti-phospho-PLK Europium-anti-phospho-PLK (Ser137) ULight-PKCULight-PKC Substrate (Ala25Ser) Europium anti-phospho-PKC Europium anti-phospho-PKC Peptide (Ala25Ser) ULight-IkBaULight-IkappaB-alpha (Ser32/36) Peptide Europium-anti-phospho-IkBa Europium-anti-phospho-IkappaB-alpha (Ser32/36) ULight-Histone H3 (Thr3/Ser10) ULight-Histone H3 (Thr3/Ser10) Peptide Europium-anti-phospho-Histone H3 (Ser10) Europium-anti-phospho-Histone H3 (Ser10) Europium-anti-phospho-Histone H3 (Thr3) Europium-anti-phospho-Histone H3 (Thr3 ULight-mTORULight-mTOR (Ser2448) Peptide Europium-anti phospho-mTOR Europium-anti phospho-mTOR (Ser2448) ULight-p70S6KULight-p70S6K (Thr389) Peptide Europium-anti-phospho-p70S6K Europium-anti-phospho-p70S6K (Thr389) ULight-Acetyl-CoA Carboxylase ULight-Acetyl-CoA Carboxylase (Ser79) SAMS Peptide Europium-anti-phospho-Acetyl-CoA Carboxylase Europium-anti-phospho-Acetyl-CoA Carboxylase (Ser79) ULight-Cdc25CULight-Cdc25C (Ser216) Peptide Europium-anti-phospho-(Ser) 14-3-3 binding motif Europium-anti-phospho-(Ser) 14-3-3 binding motif, mAb 4E2 ULight-4E-BP1ULight-eIF4E-binding protein 1 (Thr37/46) Peptide Europium-anti-phospho-4E-BP1 Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) ULight-rpS6ULight-40S Ribosomal Protein S6 (pSer235/Ser236) Peptide Europium-anti-phospho-rpS6 Europium-anti-phospho-40S Ribosomal Protein S6 (Ser235/236) ULight-Topo IIaULight-DNA Topoisomerase 2-alpha (Thr1342) Peptide Europium-anti-phospho-Topo IIa Europium-anti-phospho-DNA Topoisomerase 2-alpha (Thr1342) ULight-anti-GSTULight-anti-GST Europium-anti-phospho-ATF-2 Europium-anti-phospho-ATF-2 (Thr53) ATF-2 Fusion Protein Europium-anti-phospho-p38a Europium-anti-phospho-MAP Kinase p38-alpha (Thr180/Tyr182) GST-p38a (MAPK14), inactive Europium-anti-phospho-MEK1/2 Europium-anti-phospho-MEK1/2 (Ser217/221) GST-MAP2K1, inactive ULight-Glycogen Synthase ULight-Glycogen Synthase (Ser641/pSer657) Peptide Europium anti-phospho-Glycogen Synthase Europium anti-phospho-Glycogen Synthase (Ser641) 11 Catalog No. Phospho-Motif 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 TRF0131 TRF0220 RRPSYRK VTPRTPPP PRTSSFAE RRRSLLE RKGSLRQ RHDSGLDSM ARTKQTARKSTG TRTDSYSAG FLGFTYVAP RSAMSGLHL YRSPSMPEN (R/K)XXSXP DYSTTPGGTLFSTTPGTRI KRRRLpSSSLRAS FDEKTDDEDF PASVPPSPSLSRHSSPHQ(pS)ED www.perkinelmer.com/lance 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 anti-phospho 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/lance LANCE® Ultra Reagents for Tyr 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 parentheses. ULight Substrate 2 ULight Substrate 3 ABL [0.25]ULight-poly GT (13.1) ULight-poly GAT (11.1) ULight-TK (4.1) Carna Biosciences 08-001 ABL (T315I, ABL1) [0.5]ULight-poly GT (6.6) ULight-poly GAT (6.4) ULight-TK (4.8) Carna Biosciences 08-093 ACK [20]ULight-poly GT (27.3) ULight-poly GAT (27.2) ULight-TK (17.3) Carna Biosciences 08-103 ALK [4]ULight-IRS-1 (16.8) ULight-poly GT (9.8) ULight-TK (8.8) Carna Biosciences 08-105 ARG [0.5]ULight-poly GT (13.2) ULight-poly GAT (12.7) ULight-TK (10.6) Carna Biosciences 08-102 AXL [4]ULight-poly GT (9.8) ULight-poly GAT (5.6) Carna Biosciences 08-107 BLK [0.5]ULight-TK (31.0) ULight-poly GT (16.5) ULight-CDK1 (9.6) Carna Biosciences 08-164 BMX [0.5]ULight-poly GT (13.2) ULight-poly GAT (9.0) ULight-TK (7.1) Carna Biosciences 08-179 BRK [3]ULight-poly GT (17.4) ULight-poly GAT (3.0) Carna Biosciences 08-165 BTK [0.25]ULight-poly GAT (19.6) ULight-poly GT (11.8) ULight-TK (6.8) Carna Biosciences 08-080 CSK [20]ULight-TK (27.2) ULight-poly GAT (20.3) ULight-poly GT (10.4) Carna Biosciences 08-111 CTK [20]ULight-poly GAT (18.8) Kinase [Concentration, nM] ULight Substrate 1 Kinase Supplier Supplier Cat. No. ULight-poly GT (12.6) Carna Biosciences 08-012 DDR1 [0.5]ULight-poly GAT (3.0) Carna Biosciences 08-113 DDR2 [0.5]ULight-poly GAT (5.1) ULight-IRS-1 (4.9) ULight-poly GT (4.1) Carna Biosciences 08-114 EGFR [0.5]ULight-JAK-1 (19.0) ULight-TK (10.0) ULight-poly GAT (5.0) Carna Biosciences 08-115 EGFR (T790M) [0.5]ULight-poly GT (18.1) ULight-poly GAT (10.8) ULight-JAK-1 (3.9) Carna Biosciences 08-194 EphA1 [0.5]ULight-poly GT (24.1) ULight-poly GAT (11.4) ULight-TK (10.8) Carna Biosciences 08-120 EphA2 [0.25]ULight-TK (26.9) ULight-poly GT (12.3) ULight-poly GAT (7.1) Carna Biosciences 08-121 EphA3 [0.5]ULight-TK (22.4) ULight-poly GT (17.4) ULight-poly GAT (8.0) Carna Biosciences 08-122 EphA4 [0.5]ULight-TK (31.5) ULight-poly GT (12.6) ULight-poly GAT (11.3) Carna Biosciences 08-123 EphA5 [1]ULight-TK (35.5) ULight-poly GAT (21.9) ULight-JAK-1 (21.2) Carna Biosciences 08-124 EphA6 [0.5]ULight-poly GAT (15.5) ULight-poly GT (14.2) ULight-TK (5.4) Carna Biosciences 08-125 EphA7 [0.5]ULight-poly GT (22.7) ULight-TK (17.2) ULight-poly GAT (11.1) Carna Biosciences 08-126 EphA8 [0.25]ULight-TK (32.3) ULight-poly GT (16.4) ULight-poly GAT (7.8) Carna Biosciences 08-127 EphB1 [0.5]ULight-TK (29.9) ULight-poly GT (25.1) ULight-CDK1 (14.1) Carna Biosciences 08-128 EphB2 [0.25]ULight-TK (28.8) ULight-poly GT (27.9) ULight-poly GAT (13.2) Carna Biosciences 08-129 EphB3 [0.1]ULight-poly GT (29.0) ULight-TK (24.7) ULight-poly GAT (11.2) Carna Biosciences 08-130 EphB4 [0.5]ULight-TK (29.8) ULight-poly GT (13.7) ULight-CDK1 (12.6) Carna Biosciences 08-131 FAK [3]ULight-poly GT (20.0) ULight-poly GAT (13.0) Carna Biosciences 08-037 FER [0.25]ULight-TK (31) ULight-poly GAT (17.1) ULight-CDK1 (16.1) Carna Biosciences 08-139 FES [1]ULight-TK (29.4) ULight-poly GAT (19.0) ULight-poly GT (17.1) Carna Biosciences 08-140 13 www.perkinelmer.com/lance Kinase [Concentration, nM]ULight Substrate 1 ULight Substrate 2 ULight Substrate 3 Kinase Supplier Supplier Cat. No. FGFR1 [0.5]ULight-JAK-1 (30.8) ULight-TK (26.4) ULight-IRS-1 (13.5) Carna Biosciences 08-133 FGFR2 [0.25]ULight-JAK-1 (33.2) ULight-TK (27.1) ULight-IRS-1 (15.5) Carna Biosciences 08-134 FGFR3 [0.25]ULight-JAK-1 (27.6) ULight-TK (24.4) ULight-poly GT (16.0) Carna Biosciences 08-135 FGFR4 [3]ULight-JAK-1 (20.8) ULight-TK (17.7) ULight-poly GT (9.2) Carna Biosciences FGR [0.1]ULight-TK (29.3) ULight-poly GT (25.1) ULight-poly GAT (17.7) Carna Biosciences 08-166 FLT1 [0.25]ULight-poly GT (22.2) ULight-poly GAT (15.4) ULight-JAK-1 (10.0) Carna Biosciences 08-189 FLT3 [0.1]ULight-TK (25.9) ULight-JAK-1 (25.0) ULight-poly GT (20.8) Carna Biosciences 08-154 FLT4 [0.25]ULight-JAK-1 (36.9) ULight-TK (20.0) ULight-IRS-1 (11.4) Carna Biosciences 08-190 FMS (CSFR) [0.25]ULight-poly GT (14.6) ULight-JAK-1 (6.3) ULight-poly GAT (6.2) Carna Biosciences 08-155 FRK [0.5]ULight-poly GAT (22.9) ULight-poly GT (20.8) ULight-TK (9.7) Carna Biosciences 08-167 FYN [0.25]ULight-TK (25.9) ULight-CDK1 (19.7) ULight-poly GT (12.8) Carna Biosciences 08-068 HCK [0.25]ULight-TK (29.7) ULight-poly GT (21.3) ULight-poly GAT (13.2) Carna Biosciences 08-169 HER2 [10]ULight-poly GT (9.3) ULight-poly GAT (8.4) Carna Biosciences 08-166 HER4 (ERBB4) [0.1]ULight-poly GT (25.2) ULight-poly GAT (14.4) ULight-TK (3.1) Carna Biosciences 08-118 IGF1R [0.25]ULight-TK (25.1) ULight-poly GT (21.8) ULight-IRS-1 (15.2) Carna Biosciences 08-141 INSR [20]ULight-poly GAT (25.4) ULight-poly GT (18.3) ULight-TK (4.0) Carna Biosciences 08-242 IRR [0.5]ULight-poly GT (21.6) ULight-TK (19.2) ULight-IRS-1 (15.6) Carna Biosciences 08-143 ITK [0.5]ULight-poly GT (9.8) ULight-poly GAT (8.5) ULight-TK (8.4) Carna Biosciences 08-181 JAK1 [7]ULight-JAK-1 (19.5) ULight-poly GT (17.6) ULight-poly GAT (16.4) Carna Biosciences 08-144 JAK2 [1]ULight-JAK-1 (41.8) ULight-TK (35.5) ULight-IRS-1 (21.4) Carna Biosciences 08-045 JAK3 [0.5]ULight-JAK-1 (37.3) ULight-TK (16.1) ULight-poly GAT (6.9) Carna Biosciences 08-046 KDR [0.25]ULight-JAK-1 (30.8) ULight-TK (16.9) ULight-poly GT (8.6) Carna Biosciences 08-191 KIT [2]ULight-poly GAT (21.7) ULight-poly GT (13.0) ULight-JAK-1 (3.3) Carna Biosciences 08-056 LCK [0.5]ULight-poly GAT (16.5) ULight-TK (12.3) ULight-poly GT (10.4) Carna Biosciences 08-170 LTK [1]ULight-poly GAT (25.1) ULight-poly GT (21.3) ULight-TK (13.7) Carna Biosciences 08-106 LYNa [0.5]ULight-TK (32.5) ULight-poly GT (15.6) ULight-CDK1 (14.3) Carna Biosciences 08-171 LYNb [1]ULight-TK (33.1) ULight-CDK1 (24.0) ULight-poly GT (18.6) Carna Biosciences 08-172 MATK (Hyl) [1]ULight-poly GT (20.7) ULight-poly GAT (8.8) ` Invitrogen PV3370 MER [0.25]ULight-poly GT (29.2) ULight-poly GAT (15.9) ULight-TK (12.9) Carna Biosciences 08-108 MET [0.5]ULight-TK (24.2) ULight-JAK-1 (19.7) ULight-poly GT (17.0) Carna Biosciences 08-151 MUSK [1]ULight-poly GAT (10.6) ULight-JAK-1 (3.9) ULight-TK (3.1) Carna Biosciences 08-153 PDGFRa [0.5]ULight-poly GT (17.8) ULight-poly GAT (10.6) ULight-JAK-1 (9.0) Carna Biosciences 08-157 PDGFRb [0.25]ULight-poly GAT (12.5) ULight-poly GT (11.2) ULight-TK (4.7) Carna Biosciences 08-158 PTK2B (FAK2) [0.25] ULight-polyGT (19.2) ULight-poly GAT (12.3) Invitrogen PV4567 PYK2 [0.25]ULight-poly GT (15.5) ULight-poly GAT (7.7) Carna Biosciences 08-138 14 08-136 www.perkinelmer.com/lance ULight Substrate 3 Kinase Supplier Supplier Cat. No. RET [0.5]ULight-JAK-1 (23.6) ULight-TK (20.3) ULight-IRS-1 (6.9) Carna Biosciences 08-159 RON [0.25]ULight-poly GT (27.7) ULight-poly GAT (25.7) ULight-JAK-1 (4.9) Kinase [Concentration, nM]ULight Substrate 1 ULight Substrate 2 ROR1 [20]ULight-poly GT (3.4) Carna Biosciences 08-152 Carna Biosciences 08-160 ROR2 [20]ULight-poly GT (4.2) Carna Biosciences 08-161 ROS [0.25]ULight-TK (24.5) ULight-poly GT (23.8) ULight-IRS-1 (20.5) Carna Biosciences 08-163 SRC [0.5]ULight-TK (31.7) ULight-poly GAT (18.4) ULight-CDK1 (16.6) Carna Biosciences 08-173 SRM [0.5]ULight-TK (32.5) ULight-poly GAT (20.1) ULight-poly GT (18.3) Carna Biosciences 08-174 SYK [0.5]ULight-TK (24.6) ULight-JAK-1 (20.0) ULight-poly GAT (4.1) Carna Biosciences 08-176 TEC [0.5]ULight-poly GT (25.4) ULight-TK (18.6) ULight-poly GAT (15.4) Carna Biosciences 08-182 TIE2 [0.5]ULight-poly GT (19.3) ULight-TK (17.7) ULight-poly GAT (9.4) Carna Biosciences 08-185 TNK1 [1]ULight-poly GAT (15.2) ULight-poly GT (9.3) ULight-TK (6.8) Carna Biosciences 08-104 TRKA (NTRK1) [0.5]ULight-TK (23.2) ULight-poly GAT (17.5) ULight-poly GT (16.3) Carna Biosciences 08-186 TRKB (NTRK2) [0.5]ULight-TK (28.0) ULight-poly GAT (26.3) ULight-poly GT (22.9) Carna Biosciences 08-187 TRKC (NTRK3) [0.5]ULight-poly GT (34.1) ULight-poly GAT (22.3) ULight-TK (6.4) Carna Biosciences 08-188 TXK [3]ULight-poly GAT (18.9) ULight-poly GT (11.0) ULight-TK (5.9) Carna Biosciences 08-183 TYK2 [4]ULight-JAK-1 (15.2) ULight-IRS-1 (7.3) ULight-TK (7.3) Carna Biosciences 08-147 TYRO3 [0.25]ULight-poly GT (21.5) ULight-poly GAT (9.1) ULight-TK (6.1) Carna Biosciences 08-109 YES [0.25]ULight-TK (28.0) ULight-poly GT (15.3) ULight-poly GAT (14.0) Carna Biosciences 08-175 ZAP70 [4]ULight-poly GAT (15.3) ULight-poly GT (11.1) ULight-TK (7.6) Carna Biosciences 08-077 15 www.perkinelmer.com/lance 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 were sourced from commercial vendors, and from commercially available lots. 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 ©2009-2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 008999A_01 Printed in USA January 2012