Kinex™ Antibody Microarray Services

Our Kinex™ KAM Services allow our clients to have their cell and tissue lysates from their experimental model systems investigated for discovery of biomarker leads with our high content antibody microarrays. These antibody microarrays are convenient and very cost-effective tools to explore in a directed manner the expression and phosphorylation states of hundreds of key cell signalling proteins simultaneously with minute amounts of specimens. Samples suitable for analyses include cell extracts, fresh or frozen tissues and biofluids such as serum and cerebral spinal fluid. The results can provide novel and useful insights into differences in protein expression, phosphorylation and protein-protein interactions, and define antibody reagents that enable follow up on these findings with other immunological-based methods such as Western blotting, immunoprecipitation, ELISA and immunohistochemistry. Our integrated platform of well-established proteomics and bioinformatics services and proprietary technologies make the Kinex™ KAM antibody microarrays superior to any other commercially available antibody microarrays. Some of the key advantages of our antibody microarrays include highly validated antibody probes, wide coverage of cell signalling proteins and pathways, extensive follow-up services for validation, and supporting bioinformatics analyses for comparison purposes. In this section, we explain how the KAM antibody microarrays work and how best to use them the most effectively to advance your research programs.

Presently, Kinexus offers two different Kinex™ KAM antibody microarray that utilize complementary antibody microarray chips. The KAM-1325 antibody microarray features 875 phosphosite-specific antibodies (for phosphorylation) and 451 pan-specific antibodies (for expression levels of these phosphoproteins). The KAM-1150 antibody microarray uses over ~1150 pan-specific antibodies. When used together, the KAM-1325 and KAM-1150 chips permit screening of cell and tissue lysates with over 2000 non-redundant antibodies. These latest microarrays are the culmination of continuous on-going efforts to steadily improve the power and accuracy of our antibody microarrays over the last 9 years.

The antibodies on the Kinex™ KAM microarrays, which have been selected from more than 6000 different commercial antibodies from over 26 companies, have been independently tested in-house by Kinexus to identify many of the best immunological reagents available today to track important signal transduction proteins. The top 15% of these antibodies that performed well in Western blotting applications have been incorporated into our Kinex™ Antibody Microarrays. In addition, Kinexus has produced its own panel of well characterized cell signalling antibodies, many of which are incorporated into the KAM-1325 and KAM-1150 series antibody microarrays. Such cherry-picking is apparently not performed by other microarray companies, which rely only on one or two suppliers with dubious information about individual antibody performance. When our clients utilize the Kinex™ KAM antibody microarrays, upon request, we are pleased to disclose the commercial sources and in many cases, these antibodies are available directly from Kinexus at very affordable prices. Immunoblots with the antibodies sold by Kinexus are available for easy viewing on our Products website. The antibodies in our microarrays have been optimized to work in human, mouse and rat model systems, but have also been shown commonly to work in chicken, bovine, porcine, canine, rabbit, frog, sea star and many other diverse model systems. A complete listing of all the antibodies printed on the Kinex™ KAM microarrays in MS-Excel format is downloadable from this website.

Our antibodies are pin-printed and covalently immobilized on a high-quality glass surface coated with a proprietary 3-D polymer material to ensure high binding efficiency and specificity with the KAM-1325 microarray. For the KAM-1150 series microarrays, the antibodies are jet-sprayed on to epoxy slides. All of our microarrays are subjected to stringent quality control measures designed to ensure optimum antibody activity, printing consistency, and consistent intra-slide and inter-slide variability. The printing of individual antibodies on our microarrays is validated by probing with dye-labeled anti-rabbit, anti-mouse and anti-goat secondary antibodies. Each microarray also has loading and antibody controls to ensure the amount of deposited protein is consistent on all fields. The KAM-1325 antibody microarrays provide for semi-quantitative analyses of the expression and/or phosphorylation states of cell signalling proteins in two samples with duplicate measurements. The KAM-1150 antibody microarrays provide for semi-quantitative analyses of the expression (KAM-1150E) or tyrosine phosphorylation (KAM-1150PY) of cell signalling proteins in one sample with quadruplicate measurements. We also employ a normalization step to take into account any minor differences in protein loading on to our microarrays.

In our Kinex™ KAM-1325 Microarray Quantitation and Report Service, we provide a Microsoft Excel spreadsheet and include the (average) percent change from the control sample, and the percent range in error measurement, which can be used to determine which target proteins to follow up with as well as links to over 220 Kinections Pathway Maps. In internal studies with our KAM-1325 series antibody microarrays with and without chemical cleavage, we determined that the median spread between duplicate measurements with the same antibody in printed pairs was about 16% (i.e. the median range from the average of the duplicates was ±8% with a standard deviation of 1.0% from testing of 10 fields of 1326 antibody pairs per field). The frequency of flagged antibody spots dues to dust or misprinting is less than 2%. When the average of duplicate measurements of antibody pairs on each chip was determined for the same sample applied to different KAM Antibody Microarrays, we observed that the median value for the differences in the averages was ±8.1% with a standard deviation of 0.6% from testing of 4 pairs of fields. The dynamic range between the highest and lowest reproducible fluorescent dye-signals of captured lysate proteins from these Kinex™ chips can be over 8,000-fold after subtraction of the background signal.

With our Kinex™ KAM-1150E and KAM-1150PY Microarray Quantitation and Report Service, we provide a Microsoft Excel spreadsheet and include the averages of quadruplicate measurements, the percent change from the control samples, the standard deviation in error measurement, and calculation of p values using the Student T-test. We identify lead antibodies that reveal changes in protein expression or tyrosine phosphorylation that can be used in follow-up Western blotting experiments.

There are several different methodologies that can be used with Kinex™ KAM antibody microarrays. Method 1 involves the direct labelling of lysate proteins with a fluorescent dye and then incubation of the tagged proteins with the microarray for their immunocapture. Unbound lysate proteins are washed away and the microarray slide is scanned for the fluorescent signals associated with each antibody spot. A disadvantage of this approach is that proteins often reside in complexes, and the dye signal associated with an antibody spot may arise from different proteins. Methods 2 and 3, both involve fragmentation of the lysate proteins by chemical cleavage at cysteine (CCC) residues using Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 2-Nitro-5-thiocyanatobenzoic acid (NTCB). The CCC treatment dissociates protein complexes, and abolishes the activities of kinases, phosphatases, proteases and other enzymes, resulting in more stable peptide samples and preservation of protein phosphorylation. With Method 2, CCC treatment is performed at the time of homogenization of cells and tissues, whereas with Method 3, CCC is carried out at a later date, but also prior to labelling of the lysate proteins with a fluorescent dye. With Method 4, CCC occurs at the time of homogenization, but the lysate proteins are subsequently biotinylated rather than directly dye labeled. After capture of the biotinylated proteins on the microarray, the array is then probed with a dye-labeled anti-biotin antibody. Method 4 provides the lowest background signals and greatest dynamic range for detection of lysate proteins on the Kinex™ KAM-1325 and KAM-1150E slides, and it is our recommended procedure for the best results with this microarray for tracking changes in protein phosphorylation and expression with higher accuracy. For the KAM-1325-pY and KAM-1150PY analyses of total tyrosine phosphorylation of proteins, we recommend not subjecting the lysates samples to chemical cleavage. Detection of tyrosine phosphorylation of captured proteins on the microarray in this case is performed with a fluorescent dye-tagged anti-phosphotyrosine antibody called PYK that is produced by Kinexus (Cat. No. AB-PG001).

One key advantage of our antibody microarrays is that lysate samples from control and treated cells are labeled with the same tags (fluorescent dye and/or biotin) and analyzed together on the same chip at the same time. In our experience, the use of a two dye, competitive binding system, in which a control sample is labeled with a different dye from the treatment sample and the two samples are mixed and co-incubated with the same regions of the same chips, generates a higher rate of false leads. Unlike oligonucleotides such as DNA and RNA, proteins display strong individual differences in their relative affinities for dyes. It should be appreciated that this problem also significantly impacts other proteomics approaches such as DIGE 2D gel analysis where two samples that are labeled with different dyes are mixed prior to electrophoresis. Colour changes seen with spots evident on a DIGE 2D gel may not be related to differences in protein expression but rather dye binding to individual protein species. Clients should also be aware that cell signalling proteins are typically present at concentrations that are 100- to 1,000-fold lower than structural proteins and metabolic pathway enzymes. Consequently, these low abundance proteins are usually not evident on 2D gels without some type of special pre-enrichment. This is why we feel that antibody-based detection of proteins with our Kinex™ KAM antibody microarrays and our follow-up Kinetworks™ Custom Screens are superior and complementary methods to undertake broad studies of proteins for signalling network analyses. We use the dye combinations both with direct dye labelling of the lysate proteins as with Methods 1, 2 and 3, or for dye labelling of the anti-biotin antibody used in Method 4.

Since non-denatured proteins are commonly analyzed by Method 1, there is increased opportunity for false positives and false negatives due to antibody cross-reactivity and blocked epitopes in protein complexes. Many proteins reside in complexes with other proteins and antibodies, and as it is normally necessary to use non-denaturing conditions with antibody microarrays, many apparent changes in protein expressions or phosphorylations may arise from alterations in protein-protein interactions. In our internal studies with cells from different cells, tissues and species, only between 30 to 45% of the protein changes detected on a protein microarray were reproduced by immunoblotting. In addition, about 20 to 30% of the protein changes could not be validated by immunoblotting, because no detectable immunoreactive proteins were evident in these studies as the antibody microarray appears to be about 10-fold or more sensitive than standard Western blotting. It should be appreciated that this high rate of false positives is an inherent problem with all commercial antibody microarrays due to the reliance on non-denaturing conditions for immune capture of target proteins. To help reduce the number of false positives that are typically generated on a protein microarray, we have developed a chemical digestion step in which native proteins are cleaved into larger fragments by chemical cleavage at cysteine residues (CCC) with TCEP and NTBC. This fragmentation leads to dissociation of complexes, but does not destroy most of the epitopes recognized by phosphosite-antibodies. Typical enzymatic cleavage of proteins with proteases such as trypsin causes the loss of most phosphosite epitopes, since basic amino acids commonly surround phosphoserine and phosphothreonine sites. Furthermore, the chemical cleavage step permits more even dye-labelling of the target protein fragments that is much less reflective of the initial size of these proteins, which can vary by more than 10-fold. This chemical digestion step is an option to reduce the number of false positives for clients that are less interested in tracking protein-protein interactions changes in experimental model systems. We recommend that the protein-labelling step is carried with fluorescent dye or biotin after the CCC step, which is ideally performed at the time of homogenization. However, many users may wish to also observe changes in protein-protein interactions in lysate samples from specimens from humans and animals, and in this case, the CCC step should be omitted. We have determined that following the CCC step, the fragmented peptides in lysates are very stable at ambient temperate for over 2 weeks, and yield similar results to lysates that are immediately subjected to KAM-1325 analyses.

If the shipment of frozen lysate samples to Kinexus in Canada is too cost prohibitive for using of our proteomics services, we also offer an option where we can provide clients with Kinex™ KAM-1325 Antibody Microarray Kits so that they can perform the initial stages of the analyses in their own laboratory at reduced costs. Processed microarray chips can then be sent to Kinexus for free scanning and preparation of a KAM-1325 Report for a fee. Customers interested in this option should download our Kinex™ KAM-1325 Antibody Microarray Kit Information Package from our website.

The amount of protein required for the Kinex™ KAM-1325 Antibody Microarray service is 100 μg per sample at an approximate concentration of 3 mg/ml. For the KAM-1150 Antibody Microarray services, we recommended 200 µg per sample. If your samples have a higher concentration, we recommend sending it without further dilution and Kinexus will adjust the concentration as required during processing. In this case, we prefer a minimum volume of approximately 50 µl. If your samples have a lower concentrations, there are alternate steps that can be undertaken for ensuring optimum results. This includes concentrating your samples or providing additional dye-labeling reactions to your samples. Please contact a Kinexus Technical Service Representative for more information on how to proceed and the additional costs involved if your sample concentrations are low.

There are many different types of fractionations that can be performed, and the choice of lysis buffer used will vary depending on the type of fractionation you are considering to prepare. The simplest type of lysate preparation is the total cellular extract obtained as a total detergent-solubilized fraction. To obtain just the soluble cytoplasmic proteins, detergent should not be included in the homogenizing buffer. The remaining microsomal pellet obtained following ultracentrifugation after removal of the cytoplasmic supernatant fraction can be re-sonicated in homogenizing buffer with detergent and re-ultracentrifuged to obtain the detergent-soluble membrane fraction.

Total Cellular Extract:
For quantitation of total cellular levels of cell signalling proteins, 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. We also recommend inclusion of 0.05% SDS to further improve membrane protein solubilization. This is the most common type of fractionation prepared by clients and is optimal for monitoring changes in total protein expression. However, if proteins are re-distributed between cellular compartments as a consequence of a perturbation of an experimental model system, this will not be evident.

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 0.05% SDS 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 Fractionations:
At this time, we do not recommend you send samples from immunoprecipitation or antibody affinity pull-down experiments for the KinexTM KAM Antibody Microarray Services unless you consult with us first.

The optimum amount of protein recommended for the Kinex™ KAM-1325 Antibody Microarray is 100 μg per sample at a concentration of 3.0 mg/ml or higher. For the KAM-1150 Antibody Microarrays, we require 200 µg per sample. We recommend preparing extra lysate, if possible, for follow-up studies. If the concentration of the lysate is below 2.0 mg/ml concentration, the sample can be concentrated using an Amicon Ultra-0.5 Ultracel-3 Membrane Centrifugal Filter with a M.W. cut-off of 3,000 (Catalog Number: UFC500308, Millipore, Billerica, MA). For more information about how to concentrate samples, please contact a Kinexus Technical Services representative at info@kinexus.ca or call 1-866-546-3987.

It is highly recommended to use the Kinexus Lysis Buffer included with this kit for protein lysate preparation, as it has been optimized for the use with KAM Antibody Microarray as well as any follow-up services. Other lysis buffers commonly used for protein lysate preparation containing non-ionic detergents may be compatible with the KAM-Antibody Microarray. However, no lysis buffer containing Tris or reagents carrying reactive amine groups such as glycine and ammonia should be used to prepare lysates for the KAM Antibody Microarray. The Kinexus Lysis Buffer contains phosphatase inhibitors and the Lysis Buffer Cocktail contains protease inhibitors and DTT. Immediately prior to use, transfer the content of the Kinexus Lysis Buffer into the Lysis Buffer Cocktail. Invert the tube several times to make sure the contents are completely dissolved and store on ice. Prepare the cell or tissue lysates according to protocols listed below. The resulting protein lysate samples prepared must be frozen at -70°C or below after protein quantification unless they are to be immediately subjected to protein labelling and purification.

Once we have received your lysate samples at Kinexus, they will undergo extensive processing according to your specifications. To get a sense of how they might be handled, demonstration videos are also available for viewing on our company’s You-Tube Channel.

The final protein concentration of the cell/tissue samples should be approximately 3 mg/ml. Please record the actual concentration and volume of each sample on the Sample Description Form (NSDF-LY or CSDF-LY). We request ideally 200 µg of cell or tissue lysate for each sample submitted for analysis with each Kinex™ KAM-1325 antibody microarray and double this for the KAM-1150 antibody microarray. (If possible, it is also recommended to send an additional 10-15 µL aliquot of each sample specifically for the Bradford assay). However, it is possible to use as little as 25 µg of lysate protein for our analyses.

If any of our custom validation immunoblotting studies are to be performed based on the analysis of your Kinex™ results, we recommend sending additional lysate at this time to save on future shipping costs. We need ~350-500 µg of additional material for every 18 antibodies selected for validation Western blotting.

Samples should be stored in screw cap vials. The vials should be clearly labeled with an indelible marker with a unique identification number, parafilmed to protect against leakage, and put into another support structure such as a small box or a 50-ml conical or centrifuge tube to provide extra protection during shipping. All samples that have not been subjected to chemical cleavage at the time of homogenization must be shipped on dry ice. Approximately 5% of the time, it has been necessary for clients to re-send samples to Kinexus due to thawed samples at the time of arrival. This is most often due to insufficient dry ice for shipping or inadequate completion of shipping documentation. If the lysate samples have been prepared with chemical cleavage reagents at the time of cell or tissue lysis, they are stable for at least 2 weeks at room temperature and special refrigeration or freezing is not necessary during shipping.

Shipments sent within North America normally arrive at our facility the following day. Therefore, we recommend shipping from Monday to Wednesday to allow sufficient time to arrive safely at our facility in case of delays due to Customs or weather. For shipments from outside of North America, we recommend sending your package on Monday as shipments can take up to 5 days to arrive depending on location. You should pack enough dry ice to last a minimum of 3 days in transit (for within North America) or 5 days (for outside of North America) and preferably use large dry ice chunks mixed with nuggets to fill in the extra spaces. Dry ice sublimes at a rate of 10 to 30% (or 5-10 pounds) every 24 hours depending on the thickness of the Styrofoam container used and the size and weight of the dry ice. Pack the dry ice just before shipping to help preserve its shelf-life. Appropriate dry ice labels must be placed on the outside of the box and the weight of dry ice in kilograms written inside the label.

The aforementioned procedure has been designed to reduce the use of shipping materials and courier costs, and to ensure that your precious samples arrive in a safe and stable form at our laboratory facilities. Note that clients are responsible for payment of courier costs. Frozen sample vials should be sent to the address listed below by any express courier that accepts dry ice shipments. Cysteine chemically cleaved samples do not need to be frozen or refrigerated during shipping. We recommend Federal Express for shipments originating in North America, and World Express is the preferred courier choice outside of North America. Ship the samples to the following address and e-mail info@kinexus.ca with the courier details so we can track your package for you while it is in transit:

Kinex™ Screening Services
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

Fillable MS-Word versions of our forms are directly downloadable from the Kinexus website or can be e-mailed to you by request. Customers are required to complete the following forms for each order placed. The forms can be printed and included with your samples.

The Kinex™ KAM services permit our clients to move from “pixels” to “pathways”. As part of our KAM Antibody Microarray services, Kinexus quantifies the intensities of dye-bound proteins captured on the KAM Antibody Microarray, and we use our proprietary software to average the intensities recorded for each pair of antibody spots to calculate the differences between the control and treated lysate samples. For the KAM-1325 antibody microarray analyses this includes calculations of percent changes from control (%CFC). This permits the identification of the most promising biomarkers for further validation by immunoblotting. Direct links are provided to our Kinections Pathway Maps with over 200 local signalling network maps for many of the proteins and phosphosites tracked on the KAM microarrays. The Kinections Pathway Maps provides direct linkage of subsets of the KAM microarray results with over 200 local signalling network maps for many of the proteins and phosphosites tracked on the KAM microarray. The Report is in PDF and MS-Excel formats. The Kinections Maps may also be freely downloaded in MS PowerPoint format from the www.kinasenet.ca and the www.phosphonet.ca websites. In the MS PowerPoint, these pathways can be custom tailored for the specific needs of the users.

To provide a sense of the typical performance of individual antibodies on the Kinex™ KAM-1325 antibody microarrays and enable comparison of the specific results obtained with a tested customer cell/tissue lysate, our Analysis Report also includes summary data obtained from the analyses of many other different cell or tissue lysates samples with chemical cleavage. This includes the minimum, maximum, average, median and standard deviation values of the globally normalized signal intensities across these other studies. It also indicates which antibodies printed on the KAM-1325 chips can be ordered directly from Kinexus for follow up to experimentally validate key leads from the antibody microarray analyses.

For the KAM-1150 antibody microarray analyses, the report is provided in a Microsoft Excel spreadsheet and include the averages of quadruplicate measurements, the percent change from the control samples, the standard deviation in error measurement, and calculation of p values using the Student T-test. We then identify lead antibodies that reveal changes in protein expression or tyrosine phosphorylation that can be used in follow-up Western blotting experiments.

Kinexus offers the Kinex™ services at different pricing levels depending on the level of confidentiality required for your samples. With the full analysis with 1326 pan- and phospho-site-specific antibodies and full confidentiality, our regular price for the Kinex™ KAM-1325 Antibody Microarray Services starts at US $2,996 per slide for each pair of samples submitted and analyzed in duplicate. At this pricing level, only the species needs to be disclosed. To receive a further 35% discount off these prices, Kinexus requires the Non-Confidential Sample Description Form (NSDF-LY) to be completed in full including species, organ, tissue, cell, cell state, fractionation, perturbation, and treatment for each sample being analyzed. With the full analysis with ~1150 pan-specific antibodies and full confidentiality, our regular price for the KinexTM KAM-1150 Antibody Microarray Services starts at US $1,498 per slide for each cell or tissue lysate sample submitted and analyzed in quadruplicate. At this pricing level, only the species needs to be disclosed. To receive a further 34% discount off of these prices, Kinexus requires the Non-Confidential Sample Description Form (NSDF-LY) to be completed in full including species, organ, tissue, cell, cell state, fractionation, perturbation, and treatment for each sample being analyzed. The philosophy behind the non-confidential data pricing is to accelerate signal transduction research and knowledge within the scientific community. After a one year hold, Kinexus is permitted to post the results of a Non-Confidential analyses on its KiNET-AM website. Please note that at any time, clients can change the status of their order from Non-Confidential to Confidential by paying the difference in price. To receive a quotation or for a volume discount on large orders, please contact the Director of Sales & Marketing at 1-866-KINEXUS or 1-604-323-2547 (Extension 11) or e-mail sales@kinexus.ca .

Kinexus also offers our custom KiNetscape Network Mapping service to connect the leads from our Kinex™ KAM-1325 analyses into protein phosphorylation network maps. We have produced a database of over 16,000 experimentally confirmed kinase-substrate relationships (KSR’s), for which a specific protein kinase phosphorylates a specific phosphosite in a substrate protein in a KSR. For most of these KSR’s, the functional consequence of the phosphorylation is known or highly predictable. These KSR’s are available for viewing in the KinaseNET website. For those KSR entries from the KinaseNET database where the effects of a treatment on cells or animals generate significant changes from the antibody microarray analyses, we use the Cytoscape 3.4 program (The Cytoscape Consortium) with our customized settings to rapidly create publishable phosphorylation network maps. Figure 5 shows an example of a portion of a qualitative KiNetscape map. Custom qualitative KiNETscape maps are priced at US$250 each. A range of colour schemes are available with this graphics service. Clients should directly contact Kinexus for details if they wish to utilize this service.