Custom Kinase-Substrate Profiling (CKSP) Service

The vast majority of the proteins phosphorylated by specific protein kinases in humans and other species remain unknown despite more than 40 years of intense effort. However, with the emergence of protein kinases as one of the most promising families of drug targets in the pharmaceutical industry today, it is critical to define the proteins that are controlled by these important regulatory enzymes. Our Custom Kinase Substrate Profiling Service uniquely permits the rapid simultaneous identification of a panel of physiological substrates for most known protein kinases in any tissues or cells of specific interest. Our cost effective strategy also provides for the identification of the specific sites at which these substrates are phosphorylated, as well as commercial antibodies that can be used to specifically track and quantify these phosphosites in follow up studies. Our methodology is superior to any other procedures that have been applied to kinase substrate identification including mass spectrometry and peptide and protein microarrays, which are more expensive to perform and do not define reagents for follow on experiments. With our flexible and convenient protein kinase substrate identification service, clients can send us their own kinases and cell/tissue lysates or they can choose from our collections of over 360 active human kinases, and lysates from 14 of the most commonly studied human tumour cell lines. Within four weeks of receipt of a client order, Kinexus will provide information about a large panel of potential substrates as revealed with a 270+ phosphosite antibody microarray and validation studies with 18 of the most promising leads by immunoblotting. The selection of the candidate phosphosites for the Western blotting exploits proprietary information that Kinexus has developed to determine negative and positive determinants for specific kinase substrate recognition. The results from this unique service can be used to define signalling pathways impacted by specific kinases, identify reagents to enable kinase inhibitor discovery in vitro, and ascertain the effectiveness and specificity of kinase inhibitor drugs in living systems.

The PhosphoNET Knowledgebase available on-line from Kinexus features more than 93,000 phosphosites in over 14,000 human proteins. However, it appears that over 500,000 phosphosites are likely to exist, and with 515 known human protein kinases, each kinase appears to target about 1000 phosphosites on average. At this juncture, for more than half of the human protein kinases, not a single physiological substrate has been reported in the scientific literature. Furthermore, for about a third of the remaining human protein kinases, only one or two phosphoprotein substrates have been identified. A comprehensive understanding of the composition and architecture of cell signalling networks will require a much deeper knowledge of which phosphoproteins and which of their phosphosites are targeted by specific protein kinases, and what are the physiological consequences of their phosphorylation.

Most of the known phosphorylation sites have been elucidated through tandem MS-MS mass spectrometry. This powerful and sensitive method relies on phosphoprotein enrichment techniques such as strong cation exchange chromatography and can permit identification of hundreds of phosphosites at once. However, this method is not very quantitative, and it is impractical and costly for routine analysis of phosphorylation changes in tissues and cells that are often limiting in quantity. Moreover, with a novel phosphorylation site defined by mass spectrometry it can take more than 6 months at high expense to develop a reliable phosphosite-specific antibody for its detection by immunoblotting and immunohistochemistry. With the availability of several hundred commercial antibodies from several vendors, Kinexus has reversed the paradigm for phosphosite discovery with its Kinex™ antibody microarray and Kinetworks™ multi-immunoblotting services. With these methodologies, Kinexus uses panels of phosphosite antibodies to detect known and cross-reactive proteins that display altered phosphorylation in living systems in response to hormonal and pharmacological manipulations or, as with our Custom Kinase Substrate Profiling Service, increased phosphorylation in vitro with purified kinases. Any cross-reactive proteins can be easily enriched with the detecting phosphosite antibody and identified by MALDI mass spectrometry. Moreover, since the phosphosite epitope of the detection antibody is known, it is often easy to locate the affected phosphorylation site without further sequencing. Thus antibody-driven phosphoprotein discovery is highly cost effective, informative and enabling for immediate follow up analyses with other methodologies that exploit antibodies.

Previous services for kinase substrate identification from other companies have relied on the radioactive tagging of known purified proteins or unknown proteins in fractionated cell and tissue lysates with purified protein kinases and [gamma-32P]ATP. Enrichment of phosphoprotein substrates often involves 1D sodium dodecylsulphate gel electrophoresis (SDS-PAGE) or 2D gel electrophoresis. However, these methodologies have severe limitations with respect to separation capacity and the amount of starting material that can be resolved. To identify any specific phosphosites on purified phosphoproteins, subsequent sequencing is performed by Edman protein sequencing or tandem MS-MS mass spectrometry. As these procedures involve many steps, the risks for artifacts and loss of phosphorylation are very high. Moreover, in view of the required labour and expensive equipment required for these approaches, these strategies are beyond the financial means of most research laboratories. With our Custom Kinase Substrate Profiling Service, for their favourite experimental model system and a kinase supplied by Kinexus, any researcher can have a panel of physiological substrates, the phosphosites, and detecting phosphosite antibodies identified for less than US$3000 with our non-confidential pricing.

The Custom Kinase Substrate Profiling (CKSP) Service uses the Kinex™ Antibody Microarray (KAM) to detect the phosphorylation of hundreds of proteins that are present in crude cell and tissue lysates with commercial phosphosite antibodies. The latest version of KAM features over 270 phosphosite antibodies. Clients can provide frozen lysates of cells or tissues for kinase substrate profiling or for an added fee they can select from lysates from the Kinexus inventory, which have been prepared from the following 14 human tumour cell lines.

These cell lines have been selected on the basis of their broad representation of human tissues and cells, gender and age, and because they are commonly studied as revealed in the scientific literature. All of these cells lines can be sourced from the American Type Culture Collection for follow up studies. In addition, most of these cell lines have been treated in house at Kinexus with diverse drugs and hormones in time course and dose response studies, and they are available with the Kinex™ Reverse Lysate Microarray Service that is also offered through Kinexus. Consequently, any phosphoprotein that is identified with our CKSP service can be rapidly screened for its regulation in a broad range of other cell lines as well as monkey, rat, mouse and frog tissues with our Kinex™ Reverse Lysate Microarray Service, and further confirmed with our Custom Kinetworks™ Immunoblotting Service with the very same lysates. Moreover, interesting phosphoproteins can be screened in silico on-line with our KiNET Database with the quantitative results from over 5000 diverse cell and tissue samples analyzed with our Standard Kinetworks™ Immunoblotting Services. This ability to validate and correlate the findings from our various proteomics services is a unique and distinguishing feature of our powerful platform of integrated proteomics and bioinformatics services.

Clients are able to provide their own preparations of purified protein kinases for use with the CKSP service. Alternatively, for a fee, clients can choose from our growing inventory of over 360 active, human protein kinases as well as more than 60 additional mutant forms of these kinases. Kinexus sources these protein kinases from other vendors, and the added fee is based on our cost recovery. Consequently, the sliding price scale for these enzymes reflects the purchase price of these kinases by Kinexus. We are pleased to identify the commercial source of each protein kinase that we use upon request.

To identify kinase substrates in cell and tissue lysates, these lysates are incubated in solution the presence of ATP with (treated) and without (control) the purified protein kinase of interest. At the conclusion of the reaction, a portion of the control and treated lysates are subjected to Kinex™ Antibody Microarray analysis and tested for the increased detection of signal of fluorescent dye-labeled lysate proteins to the capture phospho-antibodies on the microarray. It is presumed that the enhancement of a phosphosite antibody signal on the KAM microarray is due to increased phosphorylation of the target phosphoprotein by the added protein kinase. However, there are other alternative explanations for elevated phosphorylation signals including cross-reactivity with other phosphoproteins, indirect phosphorylation via endogenous protein kinases that are activated by the added kinase, increased protein-protein interactions, and increased protein stability. Consequently, it is critical to confirm protein kinase substrate leads by Western blotting.

Each phosphosite antibody used on the Kinex™ Antibody Microarray has been affinity-purified against a defined phosphorylation site peptide sequence or is a monoclonal antibody. Kinexus uses information about the amino acid sequences of these phosphorylation sites to prioritize those that best match the known consensus recognition sequences of the protein kinase under study. Through bioinformatics, Kinexus has carefully examined hundreds of protein kinases to define positive and negative determinants for substrate recognition by each of these enzymes. With the CKSP Service offered by Kinexus, those phosphosites antibodies that display sequences that best match the target kinase consensus sequence are considered for further analysis by immunoblotting.

In addition to the criteria of featuring appropriate kinase recognition concensus sequences for selection of phosphosite antibodies for immunoblotting validation, Kinexus also focuses on those phosphosite antibodies that reveal the strong signals and largest increases in phosphorylation in the presence of the added protein kinase. The most promising 18 antibodies are selected for immunoblotting both the control and kinase-treated lysates. Images of these immunoblots are provided to clients, and upon request Kinexus is pleased to reveal the commercial sources of these antibodies.

The immunoblotting results can also reveal novel cross-reactive phosphoproteins that serve as protein kinase substrates. Such proteins can usually be identified following immunoprecipitation, resolution by SDS-PAGE and MALDI mass spectrometry. If clients require help for identification of intriguing cross-reactive proteins, Kinexus is pleased to assist. Clients should enquire with our technical service representatives for the costs for custom services for identification of cross-reactive proteins by mass spectrometry.

Clients have the option to use their own kinases or cell and tissues lysates, or to source these from Kinexus. In addition to some of the previously mentioned services, we also offer several other supporting services that permit our clients to further follow up on their results. These include our custom Kinetworks™ Immunoblotting Services, our IHC Immunohistochemistry Services, and our Custom Graphics Services. Our Custom Kinetworks™ KCSS 1.0 Service allows clients to choose any 3 target proteins (of different molecular weight) to be quantified for phosphorylation changes in 8 different samples side-by-side on the same immunoblot. Our technical service representatives are pleased to discuss how you can best take advantage of your results from our various services in the most cost effective way.


We plan to share the results of our Custom Kinase Substrate Profiling analyses with other scientists in our KiNET DataBank and SigNET Knowledgebank. For these rights, we have discounted our standard charges by 40% with our Non-Confidential Pricing option. The data available in KiNET and SigNET should prove to be very useful for your own reference at a later date when you compare it with your own findings using our proteomics services. Should you have any questions or concerns, we would be pleased to hear from you.

The amount of protein required for the Kinex™ Antibody Microarray (KABM) service is 100 μg per sample at a minimum concentration of 2 mg/ml (please adjust concentration accordingly with lysis buffer). The samples must be frozen and shipped to Kinexus on dry ice fresh after protein quantification without any SDS-PAGE sample buffer as the proteins are to remain in their native structure and nondenatured. The cell pellet or tissue should be homogenized in the following ice-cold lysis buffer and the final pH of the lysis buffer should be adjusted to 7.2.

Important Note: Do not add if you intend to first prepare a cytosolic fraction;
1 mM dithiothreitol (to disrupt disulfate bonds).
Important Note: dithiothreitol must be added to lysis buffer immediately before use.

NOTE: Other lysis buffers commonly used for protein lysate preparation with non-ionic detergents should still be compatible with the service, but any buffers containing Tris or reagents carrying reactive amine groups will NOT be acceptable alternatives. Please contact a Kinexus Technical Sales Representative for more information on the appropriate types of lysis buffers to use for the KinexTM Antibody Microarray Services or to request to have an aliquot of our lysis buffer for free if you can provide a courier account number to charge for the shipping costs. Our lysis buffer contains components 1-5, including phosphatase inhibitors (components 2-5) but no protease inhibitors and dithiothreitol (components 7-11). Clients must add their own dithiothreitol and protease inhibitors to the lysis buffer immediately before use. For convenience, they may choose to use the Roche Complete, Mini inhibitor cocktail tablet with the addition of pepstatin A as opposed to individual protease inhibitors.

Total cellular fractionation: For preparation of total cellular lysates, lysis and homogenization should be performed in the presence of a non-ionic detergent. We recommend the use of 1% Triton X-100 or 1% Nonidet P40, but comparable detergents are acceptable.

Subcellular fractionation: Detergents should be omitted from the homogenization buffer if the subcellular distribution of cell signalling proteins is to be examined. If a particulate-solubilized fraction is to be analyzed, a microsomal pellet should be obtained following the initial homogenization and ultracentrifugation in the absence of detergent and subsequent removal of the cytosolic supernatant. In this instance, the cytosolic extract should be removed and the microsomal pellet should then be resuspended in the homogenization buffer containing 1% Triton X-100 or 1% Nonidet P-40 and subjected to homogenization and ultracentrifugation once again. The resulting detergent-solubilized microsomal fraction should be removed and immediately assayed for its protein concentration.

Other fractionation: We do not recommend that you send samples from immunoprecipitation or antibody affinity pull-down experiments for the Custom Kinase Substrate Profiling Service.

Important things to remember are that the cells or tissues should be processed quickly at 4°C or less. Homogenization should not be performed in too large a volume to obtain lysates at the concentration required. The detergent-soluble fraction should be obtained as quickly as possible after the cells or tissues are homogenized. Sonication is required and cannot be omitted. The highest centrifugal forces available should be used to generate the detergent-soluble fraction. The supernatants should be frozen as quickly as possible if a protein assay cannot be performed immediately.

Use 1 ml of lysis buffer per 250 mg wet weight of the chopped tissue;

Rinse the tissue pieces in ice-cold PBS three times to remove blood contaminants;

Homogenize the tissue on ice with 15 strokes of a glass dounce (or 3 times for 15 seconds each time with a Brinkman Polytron Homogenizer or with a French Press as alternatives);

Sonciate the homogenate 4 times for 10 seconds on ice each time to shear nuclear DNA;

Centrifuge the homogenate at 90,000 x g or higher for 30 min at 4°C in a Beckman Table Top TL-100 ultracentrifuge, Beckman Airfuge or equivalent;

Transfer the resulting supernatant fraction to a new tube and subject it to protein assay. Using a commercial Bradford assay (available from Bio-Rad, catalogue number 500-0201) or using the standard protocol of Bradford (Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72:248-254). Bovine serum albumin should be used as the protein standard.

The final protein concentration of the cell/tissue samples should be a minimum of 2 mg/ml. If you are unable to achieve that concentration, please contact a customer service representative for assistance and suggestions. Please record the actual concentration and volume of each sample on the Sample Description Form (CKSP-NSDF-01 or CKSP-CSDF-01). Kinexus requests 1000 μg of cell or tissue lysate for each sample submitted for analysis with the Custom Kinase Substrate Profiling Service. Samples should be stored in screw cap vials. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the Sample Description Form), parafilmed, and then put into another support structure such as a 50-ml conical or centrifuge tube to provide extra protection during shipping. All lysate samples must be shipped on dry ice.

With our Custom Kinase Substrate Profiling Service, many cell lysates and a wide selection of purified and active protein kinases are available as part of this very convenient and cost effective service package. However, we do provide the option for our clients to submit their own protein kinases for use in this analysis in addition to those offered by Kinexus. In this event, clients should complete the Client Supplied Non-Confidential Kinase Description Form (CKSP-NKDF-01) provided in our client information package for these services. If you do not wish to provide the requested information, then a Client Supplied Confidential Kinase Description Form (CKSP-CKDF-01) must be completed, and full confidential pricing charges will be applied. We recognize that not all of the requested information about commercial protein kinases may be available to our clients, so please provide as much information as you are able to qualify for non-confidential pricing.

We recommend that the client supplied protein kinases are provided as concentrated as possible, preferably at around 10.1 mg/ml and within screw cap vials. We need approximately 10 µg of most protein kinases. As there are large variations in the phosphotransferase activity of protein kinases, and every kinase is unique, and we may be able to perform the Custom Kinase Substrate Profiling Service with less kinase if it still works well at a lower concentration. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the CKSP-NKDF-01 or CKSP-CKDF-01 forms), parafilmed, and then put into another support structure such as a 50-ml conical or centrifuge tube to provide extra protection during shipping. All kinase samples must be shipped on dry ice.

The aforementioned procedure has been designed to reduce the use of shipping materials and courier costs, and to ensure that your precious lysate and kinase samples arrive in a safe and stable form at our laboratory facilities. Note that clients are responsible for payment of courier costs. The vials should be sent to the address listed below by any express courier. We recommend Federal Express for shipments originating in North America, and World Express is the preferred courier choice outside of North America. Ship the cell/tissue lysates and purified protein kinases to the following address:

Kinexus Bioinformatics Corporation
Suite 1, 8755 Ash Street
Vancouver, B.C. Canada V6P 6T3
Telephone: (604) 323-2547
Facsimile: (604) 323-2548
E-mail info@kinexus.ca

Please ensure 3 copies of a signed commercial invoice (available near the end of this customer information package) accompany your shipment which specifies your lysate and kinase samples are non hazardous and non infectious. Since the protein samples are not for resale, the value of your shipment should be priced at approximately $1.00 per sample. It is highly recommended that customers e-mail their courier airway bill number and the date of departure to info@kinexus.ca so we can track your shipment in transit and ensure it arrives in a timely manner. We will send a confirmation e-mail once your shipment arrives at our facility.

Kinexus offers the Custom Kinase Substrate Profiling Service at two different pricing levels depending on the level of confidentiality required. Our regular prices for the CKSP service starts at US $4496 for each kinase and lysate pair selected for analysis if sample information is to remain fully confidential. At this pricing level, if the client provides their own lysate and/or protein kinase, only partial information about these preparations need to be disclosed in the CKSP-CSDF-01 or CKSP-CKDF-01 forms. To receive a 40% discount off the regular price, or pay US $2296 for each kinase and lysate pair, Kinexus must be allowed after at least a 6 month hold to include the results in its on-line KiNET DataBank and SigNET KnowledgeBank, which will permit third parties to access the data. If the client supplies their own cell/tissue lysates and/or protein kinases for probing the microarray, then they must also complete in full a CKSP-NSDF-01 form and/or a CKSP-CKDF-01 form.

For volume discounts or quotations for large orders, please contact the Director of Sales & Marketing at 1-866-KINEXUS (or 1-604-323-2547 (Extension 11 or Option 2 on the telephone directory) or e-mail sales@kinexus.ca.

All customers are required to complete the following forms for each order placed:

If customers wish to send their own cell or tissue lysates or purified protein kinases, they must also complete and submit the following forms:

All orders should have as a minimum: 1 SOF and 1 SIF forms completed. A new Kinexus Services Agreement may not be necessary if the client has previously placed an order with Kinexus and submitted a signed Kinexus Services Agreement at that time.

Kinexus offers several services to cost-effectively follow up the results from our Custom Kinase Substrate Profiling Service. Clients can utilize our Kinetworks™ Custom KCSS 1.0 (Multi-Sample) Protein Screen for analysis of 8 different cell or tissue lysates and choose up to 3 different phosphosite antibodies (provided the molecular weights are significantly separated by SDS-PAGE). Clients can correlate their phosphosite results with hundreds of other measurements of protein phosphorylation in hundreds of different model systems using our KiNET Database. Clients can also investigate the regulation of specific phosphosites in hundreds of cell and tissue lysates with our KinexTM Reverse Lysate Microarray Service. With our IHC Immunohistochemistry Service, we can track the location of phosphorylation changes in tissue sections. For presentation and publication purposes with the results from this Custom Kinase Substrate Profiling Service, we can prepare figures and tables with our Custom Graphics Services. For more information about these services, please contact one of our customer service representatives at info@kinexus.ca.