A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.     

High-throughput peptide synthesis and peptide purification strategy at the low micromol-scale using the 96-well format.

The increasing demand for short- and medium-sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high-throughput synthesis and purification techniques at the low-micromol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96-well format. First, a micromol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96-reaction vessel block employing standard Fmoc/tBu-chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix-assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed-phase solid-phase extraction (IP-RP-SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the micromol-scale synthesis in the 96-well format with purification by IP-RP-SPE on a 96-well micro-extraction plate format. This simple, fast and parallel approach was tested on 12-mer and 15-mer peptides to map epitopes of T- and B-cell clones, respectively. Approximately 80% of all peptides were obtained at purities > 90% without purification by RP-HPLC. In summary, this novel approach has several advantages: (i) the micromol-scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP-HPLC column was not contaminated with hydrophobic impurities.     

Determination of endothelin by an immobilized receptor assay utilizing a 96-well format.

Bovine cerebellar membranes immobilized on 96-well microtiter plates provide receptors for 125I-labeled endothelin-1 as the basis for a competitive binding assay. Adsorption of the membranes to a surface does not significantly alter the ligand-receptor interaction and reduces non-specific binding to 3-7% of total binding compared to 10-20% for a filtration technique. Considerable savings in reagents are realized since assays can be performed in 100 microliter volumes with only 10-20 micrograms of membrane protein. The 96-well format allows the rapid quantitation of large numbers of samples, and the assay is especially attractive in that it utilizes readily available reagents and equipment without the need for specific antibodies. The endothelin-receptor-based assay may be used to measure conversion of big endothelin-1 to endothelin-1 in aqueous assays. Since the presence of serum does not affect this method, tissue culture medium may be directly analyzed for endothelin production by cultured cells. All three isoforms of endothelin are detected, and the specificity of the receptor is retained since fragments and precursor forms of endothelin are not recognized. In cases where multiple endothelin isoforms may be present or where specificity of binding is in question, this assay may be used in conjunction with high pressure liquid chromatography to distinguish active peptides.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

Automated expression and solubility screening of His-tagged proteins in 96-well format

A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time.     

Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity

The measurement of prostaglandin E synthase (PGES) activity is cumbersome because the product of the reaction, PGE(2), is not readily quantitated by spectral means. The activity of isolated PGES is typically determined by PGE(2) immunoassay or by high-performance liquid chromatography using radiolabeled substrate. A relatively rapid continuous spectrophotometric assay which uses 15-hydroxyprostaglandin dehydrogenase (PGDH) to couple the oxidation of the 15-hydroxy group of PGE(2) to the formation of NADH was developed. PGDH is relatively specific for PGE(2) over the substrate for the PGES reaction, PGH(2), allowing a highly reproducible assay of PGES activity to be obtained.     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

An inexpensive technique for dot blotting in a 96-well format

Dot blotting for the quantification of proteins usually requires expensive devices. Here we present an inexpensive way to facilitate dot blotting in a 96-well format. An agarose gel was used as sample reservoir, and proteins were electrophoretically transferred from the gel to a membrane. Dots produced by this technique were applied to reproducible immunoquantification of proteins such as β-actin and doublecortin.     

Expression of soluble recombinant proteins in a cell-free system using a 96-well format

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.     

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format

Dysregulation of the brain-specific tau protein kinase II (TPK II)/cdk5 is reported to play an important role in the pathogenesis of Alzheimer's disease. We report here a quantitative scintillation proximity assay (SPA), which is suitable for determining TPK II/cdk5 activity and its inhibition. It depends upon the phosphorylation of a synthetic histone-based peptide substrate (PKTPKKAKKL), which has been biotinylated at its C-terminus. When this biotinylated peptide is incubated with [γ-³³P] ATP and TPK II/cdk5 under defined assay conditions, product formation is linear with respect to time and enzyme concentration. The production of [³³P] phosphorylated peptide is inhibited in the presence of a known TPK II/cdk5 inhibitor but is unaffected in the presence of 1% DMSO. A signal-to-noise ratio of 16:1 was obtained in a 60-min assay with an intra-assay variability of <10% in the 96-well microtiter format. The TPK II/cdk5 SPA is very robust, sensitive and simple to perform.     

Automated high throughput screening for serine kinase inhibitors using a LEADseeker scintillation proximity assay in the 1536-well format

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [(33)P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [(33)P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z' factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC(50) values for both assay formats.     

Screening for soluble expression of recombinant proteins in a 96-well format

For future structural and functional genomics programs new tools will be required. The implementation of high-throughput (HTP) methods for protein production will be an essential element. Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies. Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format. Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction. In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction. The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

A 96-well dot-blot assay for carbohydrate sulfotransferases

Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). The assay proceeds by transfer of 35S-labeled sulfate from [35S]-3(')-phosphoadenosine-5(')-phosphosulfate (PAPS) to the free amino groups of de-N-sulfated heparin (NDST-1), or the 6-hydroxyl groups of N-acetylglucosamine residues linked to a polyacrylamide scaffold (HEC-GlcNAc-6-ST). The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product "dots" for subsequent quantification. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. The assay was validated by measuring K(M) values for PAPS and K(I) values for PAP, the product of sulfuryl transfer. The assay method should be useful for inhibitor screens for both enzymes. In addition, the general assay architecture should be readily applicable to high-throughput screens of other carbohydrate sulfotransferases.     

High throughput isolation of DNA and RNA in 96-well format using a paint shaker

Most steps in plant nucleic acid isolation are easily adapted to 96-well format; however, tissue disruption typically is performed on samples individually and often is the rate-limiting step in sample processing. We have found that DNA and RNA isolation from Arabidopsis tissue can be carried out in 96-well format using a paint shaker fitted with an adapter for tissue disruption.     

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.