Standardization of an Enzyme-Linked Immunosorbent Assay for the Determination of Protein Tyrosine Kinase Activity

Schraag, Bart; Staal, Gerard E.J.; Adriaansen-Slot, Sabrina S.; Salden, Martin; Rijksen, Gert

doi:10.1006/abio.1993.1262

Cited by 19 publications

(16 citation statements)

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“…The phosphorylated substrate can be probed with a phospho-specific primary antibody and an enzyme-conjugated secondary antibody to detect phosphorylation [23]. ELISA-format kinase assays are particularly suitable for medium-throughput targeted-library screens, secondary screens, and as a follow-up to medicinal chemistry [24,25].…”

Section: Introductionmentioning

confidence: 99%

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Mand

Veach

et al. 2008

Analytical Biochemistry

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Regulated phosphorylation by protein tyrosine kinases (PTKs) such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the Chronic Myeloid Leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogelimmobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semi-quantitative detection of peptide phosphorylation by recombinant cAbl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay were demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

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Section: Introductionmentioning

confidence: 99%

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Mand

Veach

et al. 2008

Analytical Biochemistry

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“…Lck peptide substrate Ala-Glu-Glu-Glu-Ile-Tyr-Glyfinal incubation with the color-developing reagents (10)(11)(12)(13)(14). Though this ELISA-type assay can be auto-Val-Leu-Phe-Ala-Lys-Lys-Lys-Lys tagged with fluorescein either at the amino (peptide A) or carboxyl mated, this method involves several washes, liquid transfers, and incubation times, and hence is very la-terminus (peptide B) was synthesized by Research Genetics.…”

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confidence: 99%

A Homogeneous, Fluorescence Polarization Assay for Src-Family Tyrosine Kinases

Seethala

Menzel

1997

Analytical Biochemistry

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“…The extent of phosphorylation is quantified by using a suitable peroxidase substrate. This method can be standardized by the incorporation of both internal and external standards [13]. The results of these methods correlated well with the conventional radioactive assays.…”

Section: Introductionmentioning

confidence: 88%

“…In further experiments a phosphorylation time of 30 minutes was used. Detection of phosphorylated tyrosine residues was performed as described [13] by incubation with monoclonal antiphosphotyrosine antibody, rabbit anti-mouse peroxidase conjugate, and peroxidase substrate, respectively.…”

Section: Determination Of Src-like Kinase Activities By Elisamentioning

confidence: 99%

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An enzyme-linked immunosorbent assay for the determination of src-family tyrosine kinase activity in breast cancer

Rijksen

Adriaansen-Slot

Staal

1996

Breast Cancer Res Tr

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An enzyme-linked immunosorbent assay is described for the determination of protein tyrosine kinase activity originating from the presence of src-like tyrosine kinases in biological samples. In this assay a peptide derived from p34cdc2, cdc2(6-20)NH2, is coupled to the wells of a maleic anhydride-activated microtiter plate. This particular peptide has been described as an efficient and specific substrate for protein tyrosine kinases belonging to the src family kinases (Cheng et al., J.Biol.Chem. 267 (1992) 9248-9256). After incubation of the coated substrate with sample and ATP, the amount of phosphorylated tyrosyl residues is determined with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay appears to be very sensitive and is linear with sample protein concentration and phosphorylation time. Intra-assay variation is < 5%, whereas day-to-day variation is < 10%. The results of the assay have been compared with an ELISA in which the broad-specificity tyrosine kinase substrate poly(GluNa,Tyr)4:1 was coated. The results of both assays in 27 cytosolic breast cancer samples correlated very well (r = 0.94), in accordance with the predominant expression of src kinase activity in breast cancers (Ottenhoff-Kalff et al., Cancer Res. 52 (1992), 4773-4778). The present assay provides an easy, reproducible, and quick alternative for the usual radioactive methods used for the determination of src-kinase activities including immunecomplex kinase assay and TCA-precipitation assays. It allows the determination of src-like activities in human tumors for routine diagnostic purposes.

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Standardization of an Enzyme-Linked Immunosorbent Assay for the Determination of Protein Tyrosine Kinase Activity

Cited by 19 publications

References 0 publications

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

A Homogeneous, Fluorescence Polarization Assay for Src-Family Tyrosine Kinases

An enzyme-linked immunosorbent assay for the determination of src-family tyrosine kinase activity in breast cancer

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