A chemiluminescent microtiter plate assay for sensitive detection of protein kinase activity

被引:49
作者
Lehel, C [1 ]
DanielIssakani, S [1 ]
Brasseur, M [1 ]
Strulovici, B [1 ]
机构
[1] TULARIK INC, S SAN FRANCISCO, CA 94080 USA
关键词
D O I
10.1006/abio.1996.9894
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A chemiluminescent protein kinase assay using biotinylated substrate peptides captured on a streptavidin-coated microtiter plate and monoclonal antibodies to detect their phosphorylation is described. Assay conditions were optimized and validated for sensitive measurement of protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II (CAMKII), receptor interacting protein, and src activities. The newly developed chemiluminescent assay has several advantages over currently used radioactive or colorimetric methods. It is highly sensitive at low enzyme and substrate concentrations and high, close to physiological ATP levels. It is fast, simple to perform and amenable to automation and high-throughput drug screening. The assay is also robust, exhibiting minimum interference from solvents and test substances from various sources. Overall, among the presently available methods for the detection of protein kinase activity, chemiluminescence was found to provide the highest sensitivity under conditions most closely mimicking the intracellular environment. This assay is expected to be useful in both academic and industrial laboratories, especially in identifying novel classes of protein kinase inhibitors. (C) 1997 Academic Press
引用
收藏
页码:340 / 346
页数:7
相关论文
共 35 条
  • [11] GEISER T, 1988, MACROMOLECULAR SEQUE, P199
  • [12] A NOVEL AND SIMPLE METHOD TO ASSAY THE ACTIVITY OF INDIVIDUAL PROTEIN-KINASES IN A CRUDE TISSUE-EXTRACT
    GOUELI, BS
    HSIAO, K
    TEREBA, A
    GOUELI, SA
    [J]. ANALYTICAL BIOCHEMISTRY, 1995, 225 (01) : 10 - 17
  • [13] ANALYSIS OF GLYCOPROTEINS SEPARATED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS USING LECTIN BLOTTING REVEALED BY CHEMILUMINESCENCE
    GRAVEL, P
    GOLAZ, O
    WALZER, C
    HOCHSTRASSER, DF
    TURLER, H
    BALANT, LP
    [J]. ANALYTICAL BIOCHEMISTRY, 1994, 221 (01) : 66 - 71
  • [14] HAGIWARA M, 1987, MOL PHARMACOL, V32, P7
  • [15] ISOQUINOLINESULFONAMIDES, NOVEL AND POTENT INHIBITORS OF CYCLIC-NUCLEOTIDE DEPENDENT PROTEIN-KINASE AND PROTEIN KINASE-C
    HIDAKA, H
    INAGAKI, M
    KAWAMOTO, S
    SASAKI, Y
    [J]. BIOCHEMISTRY, 1984, 23 (21) : 5036 - 5041
  • [16] TNF-Dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex
    Hsu, HL
    Huang, JN
    Shu, HB
    Baichwal, V
    Goeddel, DV
    [J]. IMMUNITY, 1996, 4 (04) : 387 - 396
  • [17] INAGAKI M, 1990, J BIOL CHEM, V265, P4722
  • [18] A NONRADIOACTIVE FLUORESCENT METHOD FOR MEASURING PROTEIN-KINASE-C ACTIVITY
    ISBELL, JC
    CHRISTIAN, ST
    MASHBURN, NA
    BELL, PD
    [J]. LIFE SCIENCES, 1995, 57 (18) : 1701 - 1707
  • [19] K-252 COMPOUNDS, NOVEL AND POTENT INHIBITORS OF PROTEIN-KINASE-C AND CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN-KINASES
    KASE, H
    IWAHASHI, K
    NAKANISHI, S
    MATSUDA, Y
    YAMADA, K
    TAKAHASHI, M
    MURAKATA, C
    SATO, A
    KANEKO, M
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1987, 142 (02) : 436 - 440
  • [20] KEMP BE, 1991, METHOD ENZYMOL, V200, P121