Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence
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Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence. / Martikkala, Eija; Veltel, Stefan; Kirjavainen, Jonna; Rozwandowicz-Jansen, Anita; Lamminmäki, Urpo; Hänninen, Pekka; Härmä, Harri.
In: ANAL CHEM, Vol. 83, No. 24, 15.12.2011, p. 9230-3.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Homogeneous single-label biochemical Ras activation assay using time-resolved luminescence
AU - Martikkala, Eija
AU - Veltel, Stefan
AU - Kirjavainen, Jonna
AU - Rozwandowicz-Jansen, Anita
AU - Lamminmäki, Urpo
AU - Hänninen, Pekka
AU - Härmä, Harri
PY - 2011/12/15
Y1 - 2011/12/15
N2 - Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.
AB - Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.
KW - Energy Transfer
KW - Guanine
KW - Guanosine Triphosphate
KW - High-Throughput Screening Assays
KW - Luminescent Measurements
KW - Terbium
KW - ras Proteins
U2 - 10.1021/ac202723h
DO - 10.1021/ac202723h
M3 - SCORING: Journal article
C2 - 22098697
VL - 83
SP - 9230
EP - 9233
JO - ANAL CHEM
JF - ANAL CHEM
SN - 0003-2700
IS - 24
ER -