Technical Bulletin

LC Sciences provides a comprehensive kinase analysis service utilizing high density protein kinase substrate (PKS) peptide microarrays based on PepArray™ technology for proteomic scale kinase profiling, quantitative measurement of kinase kinetic activities, and drug discovery research. A proprietary microarray platform and advanced microfluidic technologies ensure high quality array synthesis and quantitative measurements of enzymatic reactions.

Comprehensive Service

This is a comprehensive kinase analysis service. Just send us your kinase sample (or we can order from a supplier) and we’ll perform all the necessary functions from sample assay to data analysis.

Our Kinase Profiling comprehensive service includes:

  • Synthesis of a standard content or custom designed protein kinase substrate (PKS) – peptide microarray on a PepArray™ microfluidics chip
  • Pre-staining the chip to assess synthesis quality and obtain baseline signal before the kinase sample is applied
  • Quality testing of the received kinase sample
  • On-chip enzymatic reactions
  • Specific staining of the phosphate group only on the phosphopeptides by fluorescent dye
  • Array image scan and data extraction
  • Data analysis – Basic data analysis (background subtraction, control and reference signal guided data processing, list of detected signals, data averaging, results summarized in a report) is included with our array service. Optional in-depth analysis based on your specific application is available.

High Throughput Microarray Format

Traditional kinase assays focus on a single to a few peptide or protein substrates. Profiling kinases on a peptide microarray offers the opportunity to study thousands (up to thousands of peptides/chip) of specific substrates in a single experiment. One experiment using our protein kinase substrate (PKS) – peptide microarray is equivalent to 41 experiments using a 96-well plate.

Microfluidic Technology

  • A proprietary PepArray™ microfluidics chip is used.
  • The microarray chip consists of thousands of three-dimensional chambers and is a closed system.
  • Dye oxidation and deterioration are not an issue!
  • The microfluidic technology produces a uniform distribution of the sample solutions on the array and enhances binding reactions and stringency wash processes.
  • Experiments have well-controlled temperature and flow rate and the reaction conditions are reproducible.
  • The microfluidics assay consumes as little as 70µl of total sample and is highly sensitive.

In situ Synthesis

  • In situ peptide synthesis using PGA (photogenerated acid) coupled with conventional t-Boc chemistry means high peptide quality, tight process control, and complete content flexibility.
  • Our advanced manufacturing process ensures highly uniform spots and high reproducibility across lots of arrays and yet permits total customization of contents on each individual array.
  • In comparison, spotted microarrays tend to suffer from poor spot uniformity and large spot to spot and array to array variations, which lead to large data deviations.
  • The spotting process requires significant up-front investment for peptide libraries and spotting equipment and permits no flexibility for content update or customization.

Bioinformatics

  • We have a comprehensive set of bioinformatics tools and extensive experience in microarray work.
  • We will work with you from peptide selection, array design, experiment, to data analysis.
  • We understand that what you need is to obtain reliable and rich information that is not obtainable with a single kinase assay or even a 96-well format assay.

Custom Microarray Content

These are not spotted arrays! Our PKS- peptide microarray is built on a PepArray™ microfluidics chip peptide chip. That means we can offer you standard sequence content or synthesize a custom microarray specifically designed for your experiment delivering results that cannot be achieved with an off-the-shelf assay.

Customize one of our standard microarrays or design your own PKS- Peptide Microarray. We can synthesize a completely custom microarray specifically for your experiment. Our basic format is4K peptides sequences per chip, but additional length and sequence density arrays are possible. Ask us for assistance with design of your PKS- Peptide Microarray.

Quantitative Results

All assays use universal, highly phosphate-specific fluorescent dye staining – two color experiments are possible. Well-designed negative and positive control references ensure reliable and quantifiable results. In situ synthesized PKS – peptide microarrays using our PepArray™ technology deliver more uniform spots and produce a more reliable reading of the assay signals. Multiple replicates of each peptide sequence also contributes to data confidence.

Sensitive and Safe Detection

All assays use universal, highly phosphate-specific fluorescent dye staining – two color experiments are possible. There is no need for radioisotope labeling or for antibody binding. This eliminates potential signal bias due to sequence specificity of antibodies and loss of signal due to lack of suitable detection antibody. All PKS – peptide microarray chips are pre-stained to assess synthesis quality and obtain reliable baseline phosphorylation signal before the kinase sample is applied. Detected signals are derived from observed and reference signals; non-specific protein interaction signals are subtracted. (Antibody-based detection could also apply on your request.)

Unique Signal References

Well-designed negative and positive control references ensure reliable and quantifiable results. LC Sciences has been a pioneer in the use of phosphopeptides as positive control references on protein kinase substrate microarrays. These controls are non-peptide sequences or pS/pT/pY and are highly sensitive. Test sequence signals can be directly compared to phosphopeptide signals. We employ established microarray analysis software to extract detectable signals after non-linear baseline correction of quantitative calculations using reference signals and statistical analyses.

Reliable Results

In situ synthesized PKS – peptide microarrays using our PepArray™ technology deliver more uniform spots compared to spotted arrays which produce a more reliable reading of the assay signals. Microfluidic technology means experiments have well-controlled temperature and flow rate and the reaction conditions are reproducible. Multiple QC steps are implemented at various stages of our microarray manufacturing and assay processes. Multiple QC control peptides are synthesized on chip and used for monitoring synthesis and assays. Multiple replicates of each peptide sequence also contributes to data confidence.

Picoliter Scale Titer Plate Microarrays

Our unique PepArray™ technology makes it possible for us to vary the density of peptides synthesized across an array. Repeating sequences of the same peptide can be synthesized at different molecular densities and thus are presented at different “concentrations” creating a gradient across the array. A 4K feature microarray can generate the same amount of data equivalent to that of 40 conventional 96-well microtiter plates. Generate hundreds of enzymatic reaction curves with a single experiment. Make parallel measurements of multiple reaction curves to determine of VMAX and KM.


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