PepChip Kinase Products
Kinase microarray slides available as ready-to-use products or as full service
Protein phosphorylation is involved in almost all cellular processes. Protein kinases exert their functions in networks comprised of interconnected signal transduction pathways. Abnormalities in these pathways are implicated in the etiology of numerous diseases, and protein kinases are currently a main subject of research and important targets in drug discovery programs. Pepscan has therefore developed a range of Pepchip products, to assist scientists in profiling of kinase networks.
PepChip Kinomics arrays and the PepChip Kinase arrays are routinely used for kinase profiling, whereas PepChip Evaluation slides are used for optimization of the reaction conditions. The microarray slides are available as ready-to-use products or as service in which we perform the kinase experiments for you.
|Product||PepChip Kinomics Array||PepChip Kinase Array||Kinase Evaluation Slide|
|Substrate||Human sequences||Mixed species (bovine, pig, mouse,rat, human, etc)||Mixed species|
|Peptides||1,024 in triplicate||1,024 in triplicate||192 in duplicate|
|Quantification||PepChip Kinomics Grid.zip||PepChip Kinase Grid.zip||PepChip Trial Grid.zip|
|Substrate-list||PepChip Kinomics Map.zip||PepChip Kinase Map.zip||PepChip Trial Map.zip|
Our flagship product, the PepChip Kinomics array, contains 1,024 peptides in triplicate derived from known phosphorylation sites from human protein sequences, annotated with upstream kinases. The PepChip Kinase array contains a collection of 1,184 different peptide substrates in duplicate, from multiple species. For working with PepChip in your own laboratory, PepChip products are available as consumables. The following equipment and facilities are needed to successfully perform the experiments:
- A lab for working with radioisotopes, specifically ³³Ρ
- A cooled microfuge
- An imaging device for radioactivity, either using phosphor storage screens or direct imaging
- Microarray analysis and interpretation software
Full Service Profiling
For PepChip Kinomics Arrays, full service profiling is also available at a fee-for-service. Pepscan’s full kinase profiling service is comprehensive, requiring only a lysate, kinase protein or even cells or tissue samples. The customer ships cell or tissue lysates to Pepscan on dry ice. Typically, 20-100 micrograms of total protein lysate in 10-50 microliters is required for one array. Biological replicates are recommended. Pepscan experts take care of all experiments and analysis and deliver the results via email and download, including a summary report, raw data, slide scans and analysis files.
Advantages over other approaches
Mass spectrometry-based phospho-proteomics is complex and time-consuming and requires capital equipment. Kinase assays on individual substrates do not take into account the network-nature of regulation by phosphorylation. Profiling based on detection with phosphorylation-specific antibodies is site-based and not necessarily kinase-activity-based. It is also limited to sites and kinase activities for which antibodies are available. And finally, many antibodies lack the specificity required for application in multiplex panels. PepChip kinase profiling overcomes these shortcomings.
Protein phosphorylation is the most common post-translational modification, regulating almost all cellular processes. Protein kinases form a large gene family with more than 500 members. Kinases exert their functions in networks comprised of interconnected signal transduction pathways. Abnormalities in these pathways are implicated in the etiology of numerous diseases, including cancer and inflammatory diseases. This has motivated pharmaceutical companies to pursue protein kinases as drug targets, now accounting for a significant portion of drug discovery programs and several successful products on the market.
PepChip assay principle
PepChip Kinomics arrays are incubated with cell or tissue lysates or purified kinase protein. The assay is based on isotopic detection of phosphorylation using γ-33P-labelled ATP. The kinases are incubated on the array in a reaction mixture containing the isotopic label. Kinases transfer isotopically labeled phosphate groups to serine, threonine and tyrosine residues in the peptides on the surface of the array. Incubated PepChip arrays are imaged for radioactivity, quantified and analyzed to produce an annotated list of phosphorylation levels. Assay protocols can be optimized over a wide range of conditions. Isotopic detection is highly sensitive and not biased towards specific phosphorylation sites.
Advantages over other approaches
Mass spectrometry-based phosphoproteomics is complex and time-consuming and requires capital equipment. Kinase assays on individual substrates do not take into account the network-nature of regulation by phosphorylation. Profiling based on detection with phosphorylation-specific antibodies is site-based and not necessarily kinase-activity based. It is also limited to sites and activities for which antibodies are available. And finally, many antibodies lack the specificity required for application in multiplex panels. PepChip kinase profiling overcomes these shortcomings.
Where traditional approaches may fail
The complexity and redundancy of signal transduction networks, combined with the inherent lack of specificity of kinases and kinase inhibitors renders the description of their effects on protein kinases difficult. The traditional reductionist’s approach often does not provide a complete and accurate description of protein kinase networks. PepChip Kinase profiling provides information on a completely different level, providing a broad view of kinase activities.
PepChip Kinase profiling gives you better insight in the role of kinase proteins, networks and kinase inhibitors in disease and health. Its results help to develop better inhibitors and better tools to apply therapies successfully.
|Substrates||11-mer synthetic peptides with the phosphorylation site in position 6|
|Number||1.024 peptide substrates in triplicate|
|Array format||8×8 spot fields
4×4 fields in each replicate subarray
3 replicate subarrays 32×32 spots in each replicate subarray
|Spotting controls||16 (for production QC)|
|Grid controls||32 annotated substrates|
|Substrate source||Public - up-to-date annotated databases|
|Substrate information||HPRD_NR - ID number in www.hrpd.org (if available)
CST_NR - ID number in Phosphosite (if available)
PEPTIDE MOTIF - sequence
PROTEIN - originator protein name
MOL_CL - molecular class of originator protein
BIOL_PROC - biological process protein
PROT_ID - originator protein ID
OMIM_ID - originator protein OMIM ID
PTMS - link to post. transl. mod. In HPRD (if available)
ABBR - protein name abbreviation
PSITE - AA and AA-number in originator protein
UP-KIN - reported upstream kinases
UP_KIN_ID - reported upstream kinase IDs
CST_AB - CST antibody catalog number (if available)
|Slide format||Standard 25×75 mm glass slide|
|Slide format||Proprietary hydrophilic polymer coating|
|Coupling chemistry||Proprietary covalent coupling chemistry|
|Standard detection||³³P labeling using ƴ-³³P-ATP|
|Label required||10-30 µCi per slide|
|Incubation time||Standard 2 hours|
|Exposure time||24-96 hours on a phosphor storage screen|