Development and comparison of two nonradioactive kinase assays for I kappa B kinase

Sadler, Tammy; Achilleos, Maria; Ragunathan, Shoba; Pitkin, Adam; LaRocque, James; Morin, John M.; Annable, Rebecca; Greenberger, Lee M.; Frost, Philip; Zhang, Yixian

doi:10.1016/j.ab.2003.11.021

Cited by 21 publications

(14 citation statements)

References 26 publications

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“…We employed TR-FRET technology to develop a non-radioisotopic and robust biochemical assay, and identifi ed both a potent substrate peptide with physiological b-pFKD), we screened 87 phosphor-(Ser/Thr)-specifi c antibodies from Serine/Threonine Kinase Substrate Screening Kit (Cell Signaling Technology) with time-resolved fl uorometry (TRF, also known as DELFIA ® technology). 23 We incubated b-FKD (biotin-DELMEFSFKDQEAKV) with 50 μM ATP and 120 nM Plk1∆C in buffer A for 5 h at 25°C, and transferred the reaction mixture to a streptavidin-coated 96-well plate to immobilize b-pFKD. Then we dispensed antibodies, with one per well, and incubated the plate.…”

Section: Methodsmentioning

confidence: 99%

“…Although the titration curve lost linearity when the concentration of b-pFKD was higher than 40 nM (Fig. 4A), S/B ratio 27 was DELFIA ® technology 23 (see Materials and Methods for details). Among the 87 antibodies tested, anti-phospho-(S/T)F antibody (p(S/T)F Ab) showed the highest specifi city to b-pFKD over b-FKD (Fig.…”

Section: Assay Development Of Plk1 Tr-fret In a 384-well Plate Formatmentioning

confidence: 99%

“…20 Serine 198 residue is the phosphorylation site of Plk1 21 and a hydrophobic residue at +1 position and acidic residues at −2 and +3 positions are required for this phosphorylation. 22 Subsequently, we tested 87 antibodies in a 96-well format for the detection of the phosphorylated form of FKD using time-resolved fl uorometry technology (TRF, also known as DELFIA ® technology), 23 and found that anti-phospho-(S/T)F antibody effi ciently and specifi cally recognizes phosphorylated FKD peptide. Using FKD peptide and anti-phospho-(S/T)F antibody, we succeeded in developing a robust TR-FRET assay in a 384-well plate format.…”

mentioning

confidence: 99%

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Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

Tanaka

Koresawa

Iida

et al. 2010

ASSAY and Drug Development Technologies

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ABBREVIATIONS: % CV, percentage coeffi cient of variation; APC, allophycocyanin; b-FKD, biotinylated FKD peptide; DELFIA ® , dissociation-enhanced lanthanide fl uorescence immunoassay; b-ASFA, biotinylated ASFA peptide; DMSO, dimethyl sulfoxide; Eu-p(S/T)F Ab, anti-phosphor-(S/T)F antibody labeled with europium-chelate;FI, fl uorescence intensity; HTS, high-throughput screening; HPV16, human papillomavirus type 16; IC 50 , a half-maximal inhibitory concentration; mean + 3 std, the assay mean plus 3 times the standard deviation of percentage inhibition; Plk1∆C, polo-like kinase 1 deletion mutant with the N-terminal kinase domain; Plk1T210D, ABSTRACT One of the challenges to develop time-resolved fl uorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/ Thr) protein kinase is to select an optimal peptide substrate and a specifi c phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1

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Section: Methodsmentioning

confidence: 99%

Section: Assay Development Of Plk1 Tr-fret In a 384-well Plate Formatmentioning

confidence: 99%

mentioning

confidence: 99%

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Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

Tanaka

Koresawa

Iida

et al. 2010

ASSAY and Drug Development Technologies

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“…They were employed, among others, for the sensitive detection of DNA and proteins [78], and for a variety of enzyme assays [79][80][81]. Recently, a novel type of TRF assay based on DBO as a truly fluorescent (rather than phosphorescent) label has been introduced, thereby by-passing the need for rare earth metal ions [82].…”

Section: Applications Of Supramolecular Radiative Decay Engineeringmentioning

confidence: 99%

Squeezing Fluorescent Dyes into Nanoscale Containers—The Supramolecular Approach to Radiative Decay Engineering

Nau

Hennig

Koner

2007

Springer Series on Fluorescence

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“…These follow loss of reactants (ATP), production of products (ADP or phosphorylated substrates), or competition between test compounds and suitably labeled ligands that can bind to the active site of the kinase. [24] Specifically these include: 1) cell-based assays; [25] 2) ATP-dependent assays (colorimetric, fluorescent and bioluminescent readouts); [24] 3) antibody-based, time-resolved fluorescence resonance energy transfer (FRET) readout assays; [26] 4) radioassay methods; 5) ligand displacement assays (fluorescence polarization or FRET); [27,28] and 6) phosphospecific staining methods (AlphaScreen TM , [29] iron quenching, [30] immobilized metal ion affinity based polarization (IMAP), [31] Pro-Q TM Diamond stain [32] ).…”

Section: Introductionmentioning

confidence: 99%

An ESI‐MS/MS Method for Screening of Small‐Molecule Mixtures against Glycogen Synthase Kinase‐3β (GSK‐3β)

et al. 2008

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Glycogen synthase kinase-3beta (GSK-3beta) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-(32)P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3beta. Optimum reaction conditions (level of Mg(2+), buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3beta and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3beta activity from reaction volumes of approximately 40 microL and that it can be used to assess IC(50) values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3beta.

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Development and comparison of two nonradioactive kinase assays for I kappa B kinase

Cited by 21 publications

References 26 publications

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

Squeezing Fluorescent Dyes into Nanoscale Containers—The Supramolecular Approach to Radiative Decay Engineering

An ESI‐MS/MS Method for Screening of Small‐Molecule Mixtures against Glycogen Synthase Kinase‐3β (GSK‐3β)

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