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Kinex™ Antibody Microarray Services

Introduction

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 microarray 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-900P antibody microarray features 613 phosphosite-specific antibodies (for phosphorylation) and 265 pan-specific antibodies (for expression levels of these phosphoproteins). The KAM-1100E antibody microarray uses over 1100 pan-specific antibodies. When used together, the KAM-900P and KAM-1100E chips permit screening of cell and tissue lysates with over 1700 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-900P and KAM-1100E 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 KAP-900P microarray. For the KAM-1100 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-900P antibody microarrays provide for semi-quantitative analyses of the expression and/or phosphorylation states of cell signalling proteins in two samples. The quantitative analysis of the strength of the fluorescence signals for each captured target protein is based on duplicate 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 Microarray Quantitation and Report Service, we provide a Microsoft Excel spreadsheet and include the (average) percent change from the control sample, the percent range in error measurement, and Z-ratios that can be used to determine which target proteins to follow up with as well as a Kinections Pathway Map analysis feature. In internal studies with our KAM-900P series antibody microarrays without chemical cleavage, we determined that the median spread between duplicate measurements with the same antibody in printed pairs was about 24% (i.e. the median range from the average of the duplicates was ±12% with a standard deviation of 2.0% from testing of 12 fields of 878 antibody pairs per field). With chemical cleavage, we determined that the median spread between duplicate measurements with the same antibody in printed pairs was about 30% (i.e. the median range from the average of the duplicates was ±15% with a standard deviation of 2.0% from testing of 54 fields of 877 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 10,000-fold.

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 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.

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-900P 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-900P 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-900P Report for a fee. Customers interested in this option should download our Kinex™ KAM-900P Antibody Microarray Kit Information Package from our website.

Quantity of Lysate Required

The amount of protein required for the Kinex™ KAM-900P Antibody Microarray service is 100 μg per sample at an approximate concentration of 3 mg/ml. 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.

Lysate Buffer

The standard ingredients for our lysis buffer are listed below, however other lysis buffers commonly used for protein lysate preparation with non-ionic detergents should be compatible with the service. However any lysis buffers containing Tris or reagents carrying reactive amine groups are not acceptable alternatives. These will interfere with lysate protein labelling. Please contact Kinexus for more information on the appropriate types of lysis buffers to use or email info@kinexus.ca to request an aliquot of our lysis buffer to be sent at no cost. We only require a courier account number to cover the shipping expenses. Your cell pellets or tissues should be homogenized in ice-cold lysis buffer.

The reagents in the Kinexus Lysis Buffer (pH 7.2) include:
  1. 20 mM MOPS, pH 7.0;
  2. 2 mM EGTA (to bind calcium);
  3. 5 mM EDTA (to bind magnesium and manganese);
  4. 50 mM sodium fluoride (to inhibit protein-serine phosphatases);
  5. 60 mM beta-glycerophosphate, pH 7.2 (to inhibit protein-serine phosphatases);
  6. 25 mM sodium pyrophosphate (to inhibit protein-serine phosphatases);
  7. 2.5 mM sodium orthovanadate (to inhibit protein-tyrosine phosphatases);
  8. 50 nM phenylarsine oxide;
  9. 1% Triton X-100* (can be substituted with 1% Nonidet P-40);

*NOTE: A detergent (Triton X-100) is required for preparing total detergent-solubilized lysates. The detergent should be omitted from the lysis buffer if a subcellular fractionation is to be performed.

For chemical cleavage harvesting only:
  1. 10 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride);
  2. 100 mM NTCB (2-nitro-5-thiocyanatobenzoic acid) (added after sonication);

Protease Inhibitors and Dithiothreitol:
  1. 0.5 µM aprotinin (to inhibit proteases);
  2. 3 mM benzamidine (to inhibit proteases);
  3. 1 mM Petabloc (to inhibit proteases);
  4. 10 µM leupeptin (to inhibit proteases); and
  5. 1 mM dithiothreitol (to disrupt disulphate bonds).

The protease inhibitors and dithiothreitol (DTT) must be added to lysis buffer immediately before use and samples should be processed as quickly as possible. Not all protease inhibitors are required, but it is optimal to use as many as possible. For convenience, the Roche Complete Mini Inhibitor Cocktail tablet can be used to replace the individual protease inhibitors. If the lysate proteins are to remain in their native structure and not denatured, the chemical cleavage step should not be used, and the samples must be frozen and shipped to Kinexus on dry ice. Samples that have been subjected to chemical cleavage or homogenized directly into 1X SDS-PAGE sample buffer can be sent to Kinexus without the need for refrigeration or freeze during shipping.

Important points to remember include:
  1. The cells or tissues should be processed quickly at 4°C or less if the samples are not subjected to chemical cleavage at the time of homogenization;
  2. Add the protease inhibitors and DTT to the lysis buffer just before processing samples;
  3. Ensure the contents are completely dissolved and store on ice;
  4. Homogenization should be performed in small volumes of lysis buffer to obtain protein lysates at high concentrations, ideally at 3-4 mg/ml or higher. The concentrations can be diluted later if required;
  5. detergent-soluble fraction should be obtained as quickly as possible after the cells or tissues are homogenized;
  6. Sonication is required for optimal results (do not over sonicate);
  7. The highest centrifugal forces available should be used to generate the detergent-soluble fraction;
  8. The supernatants should be frozen as quickly as possible if a protein assay cannot be performed immediately. Lysates should be stored at -70°C, unless these have been subjected to chemical cleavage or processed in SDS-PAGE sample buffer.
  9. We recommend harvesting cells and tissues with the chemical cleavage reagents (TCEP and NTCB) to help reduce the number of false positives that can arise from the use of non-denatured proteins on the antibody microarray. If you choose to prepare samples without the chemical cleavage method, then omit the sections below outlined in red. However, you should let us know and we can include the chemical cleavage step for you prior to probing your lysates on the microarray. Note that the best results are obtained if the chemical cleavage is performed during initial lysate preparation.

Fractionations

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. 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 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.

Protein Lysate Preparation With and Without Chemical Cleavage

The optimum amount of protein recommended for the Kinex™ KAM-900P Antibody Microarray is 100 μg per sample at a concentration of 3.0 mg/ml or higher. 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.

  1. Preparation of Lysates from Cells with Chemical Cleavage
    1. Adherent Cells:
      1. Remove medium from culture dishes containing approximately 1×106 to 2×106 cells for each sample to be analyzed using the KAM-900P microarray.
      2. Rinse the cells in the dishes twice with ice-cold Phosphate Buffered Saline (PBS) to remove medium residue (serum must be completely removed) and aspirate as much PBS as possible after the last rinse.
      3. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Invert the tube several times to ensure the contents are completely dissolved and store on ice. Add 200 µl of the ice-cold Kinexus Lysis Buffer to a 150-mm culture dish, or add 100 μl ice-cold Kinexus Lysis Buffer to a 100-mm culture dish. Also, add 25 µl of 10 mM TCEP to 500 μl of lysis buffer for a final concentration of 0.5 mM TCEP. Adjust the pH of the lysis buffer containing 0.5 mM TCEP to pH 9 (approximately 2 μL of 10 N NaOH per 1 mL buffer).
      4. Scrape the cells in Kinexus Lysis Buffer, collect the resulting cell suspension from dishes and transfer it into a 1.5-ml microcentrifuge tube. Check to make sure that the pH is 9.0.
      5. Sonicate using a microprobe sonicator 4 times for 10 seconds each with 10-second intervals on ice to rupture the cells and to shear nuclear DNA. Alternatively, passing the cell suspension through a 26-gauge needle until the sample is no longer viscous is also acceptable if a sonicator is not accessible. This step is crucial and cannot be omitted. Add 6 µL of 100 mM NTCB per 100 µL cell homogenate for a final concentration of 6 mM NTCB, and make sure that the pH is 9.0 and adjust with 10 N NaOH if necessary). Incubate the homogenate at 45°C in a water bath for 30 minutes.
      6. Centrifuge the resulting lysate homogenate at 90,000 x g or above for 30 minutes at room temperature in a Beckman Table Top TL-100 ultracentrifuge, Beckman Airfuge or equivalent. Alternatively, clearing homogenates at maximum speed (15,000-17,000 rpm) on a benchtop microcentrifuge for 30 minutes at room temperature is also acceptable.
      7. Transfer the resulting supernatant to a new 1.5-ml microcentrifuge tube.
      8. Remove a small aliquot and determine its protein concentration using a commercial Bradford assay reagent (available from Bio-Rad, catalogue number 500-0201) or following 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 (BSA) is used as the protein standard. The protein concentration obtained should be approximately 3.0 mg/ml or higher. If the concentration obtained is less than 1.0 mg/ml, samples should be concentrated using an Amicon Ultra-0.5 Centrifugal Filter (Millipore).
      9. Check the pH of the lysates and adjust to pH 7.0-7.4 with 1 M HCl if necessary. Aliquot and set aside 100 μg for each lysate to be analyzed with the KAM-900P chip.
      10. If you wish to have Kinexus perform the custom immunoblotting follow-up analysis, aliquot 350-500 μg for each 18 antibodies to be tested, and boil in SDS-Sample Buffer following the protocols specified on our website. Chemically cleaved lysates are stable at ambient temperature for at least 2 weeks. Store any remaining lysates at -70°C for subsequent validation studies.
    2. Suspension Cells:
      1. Transfer cells with medium from cell culture flasks into appropriate sized tubes and centrifuge at 500 x g for 2 minutes at 4°C in a swinging bucket benchtop centrifuge. Remove as much medium from the cell pellet as possible without disrupting cells.
      2. Wash the pellet by gently resuspending the cells in ice-cold PBS, followed by centrifugation as above. Repeat this step once to ensure complete removal of serum. Remove as much PBS as possible after the final wash.
      3. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Invert the tube several times until dissolved and store on ice. Add 25 µl of 10 mM TCEP to 500 μl of lysis buffer for a final concentration of 0.5 mM TCEP, and adjust the pH to 9 (which is approximately 2 µL of 10 N NaOH per 1 mL buffer). Add an adequate amount of the ice-cold Kinexus Lysis Buffer to the sample based on the number and type of cells to achieve a final total protein concentration of approximately 3.0 mg/ml.
      4. Follow Steps # 5 through 10 as described in the Adherent Cells Section above.
  2. Preparation of Lysates from Cells without Chemical Cleavage
    1. Adherent Cells:
      1. Remove medium from culture dishes containing approximately 1×106 to 2×106 cells for each sample to be analyzed using the KAM-900P microarray.
      2. Rinse the cells in the dishes twice with ice-cold Phosphate Buffered Saline (PBS) to remove medium residue (serum must be completely removed) and aspirate as much PBS as possible after the last rinse.
      3. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Invert the tube several times to ensure the contents are completely dissolved and store on ice. Add 200 µl of the ice-cold Kinexus Lysis Buffer to a 150-mm culture dish, or add 100 µl ice-cold Kinexus Lysis Buffer to a 100-mm culture dish.
      4. Scrape the cells in Kinexus Lysis Buffer, collect the resulting cell suspension from dishes and transfer it into a 1.5-ml microcentrifuge tube.
      5. Sonicate using a microprobe sonicator 4 times for 10 seconds each with 10-second intervals on ice to rupture the cells and to shear nuclear DNA. Alternatively, passing the cell suspension through a 26-gauge needle until the sample is no longer viscous is also acceptable if a sonicator is not accessible. This step is crucial and cannot be omitted.
      6. Centrifuge the resulting lysate homogenate at 90,000 x g or above for 30 minutes at 4°C in a Beckman Table Top TL-100 ultracentrifuge, Beckman Airfuge or equivalent. Alternatively, clearing homogenates at maximum speed (15,000-17,000 rpm) on a benchtop microcentrifuge for 30 minutes at 4°C is also acceptable.
      7. Transfer the resulting supernatant to a new 1.5-ml microcentrifuge tube. The following steps should be performed as quickly as possible with the supernatant fraction kept in an ice bath.
      8. Remove a small aliquot and determine its protein concentration using a commercial Bradford assay reagent (available from Bio-Rad, catalogue number 500-0201) or following 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 (BSA) is used as the protein standard. The protein concentration obtained should be approximately 3.0 mg/ml or higher. If the concentration obtained is less than 1.0 mg/ml, samples should be concentrated using an Amicon Ultra-0.5 Centrifugal Filter (Millipore).
      9. Aliquot and set aside 100 μg for each lysate to be analyzed with the KAM-900P chip.
      10. Store any remaining lysates at -70°C for subsequent validation studies. If you wish to have Kinexus perform the custom immunoblotting follow-up analysis, aliquot 350-500 µg for each 18 antibodies to be tested, and boil in SDS-Sample Buffer following the protocols specified on our website. Label and freeze remaining lysates.
    2. Suspension Cells:
      1. Transfer cells with medium from cell culture flasks into appropriate sized tubes and centrifuge at 500 x g for 2 minutes at 4°C in a swinging bucket benchtop centrifuge. Remove as much medium from the cell pellet as possible without disrupting cells.
      2. Wash the pellet by gently resuspending the cells in ice-cold PBS, followed by centrifugation as above. Repeat this step once to ensure complete removal of serum. Remove as much PBS as possible after the final wash.
      3. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Invert the tube several times until dissolved and store on ice. Add an adequate amount of the ice-cold Kinexus Lysis Buffer to the sample based on the number and type of cells to achieve a final total protein concentration of approximately 3.0 mg/ml.
      4. Follow Steps # 5 through 10 as described in the Adherent Cells Section above.
  3. Preparation of Lysates from Tissues with Chemical Cleavage
    1. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Add 25 µl of 10 mM TCEP to 500 μl of lysis buffer for a final concentration of 0.5 mM TCEP. Invert the tube several times until dissolved and adjust the pH of the lysis buffer containing 0.5 mM to pH 9 (which is approximately 2 µL of 10 N NaOH per 1 mL buffer) and store on ice. Use approximately 1 ml of the Kinexus Lysis Buffer per 250 mg wet tissue.
    2. Cut the tissue into smaller pieces and rinse them in ice-cold PBS three times to remove any blood contaminants.
    3. Homogenize the tissue on ice with 15 strokes of a glass douncer (or 3 times for 15 seconds each time with a Brinkman Polytron Homogenizer or with a French Press as alternative).
    4. Sonicate the homogenate 4 times for 10 seconds on ice each time to shear nuclear DNA.
    5. Add 6 μL of 100 mM NTCB per 100 µL cell homogenate for a final concentration of 6 mM NTCB, and adjust the pH to 9.0 with 10 N NaOH if necessary. Incubate the homogenate at 45°C water bath for 30 minutes.
    6. Centrifuge the homogenate at 90,000 x g or higher for 30 minutes at room temperature in a Beckman Table Top TL-100 ultracentrifuge, Beckman Airfuge or equivalent. Alternatively, clients can also centrifuge at maximum speed (15,000 – 17,000 rpm) on a benchtop microcentrifuge for 30 minutes at room temperature.
    7. The following steps should be performed as quickly as possible once the supernatant fraction is obtained. Check that the pH of the lysates, which should be close to neutral (pH 7.0-7.4) and adjust with 1 M HCl if necessary.
    8. Transfer the resulting supernatant fraction to a new tube and subject it to a protein assay using a commercial Bradford assay reagent or using the standard protocol of Bradford. BSA should be used as the protein standard. The protein concentration obtained should be approximately 15-20 mg/ml or higher, but a final concentration of only 3 mg/ml for the antibody microarray is needed. If the concentration obtained is less than 1.0 mg/ml, samples should be concentrated using an Amicon Ultra-0.5 Centrifugal Filter (Millipore).
    9. Aliquot 100 μg for each lysate to be analyzed with the KAM-900P antibody microarray.
    10. Chemically cleaved lysates are stable at ambient temperature for at least 2 weeks. Store any remaining lysates at -70°C for subsequent validation studies.
  4. Preparation of Lysates from Tissues without Chemical Cleavage
    1. Mix the components in the Kinexus Lysis Buffer as listed in Section 11. Invert the tube several times until dissolved and store on ice. Use approximately 1 ml of the Kinexus Lysis Buffer per 250 mg wet tissue.
    2. Cut the tissue into smaller pieces and rinse them in ice-cold PBS three times to remove any blood contaminants.
    3. Homogenize the tissue on ice with 15 strokes of a glass douncer (or 3 times for 15 seconds each time with a Brinkman Polytron Homogenizer or with a French Press as alternative).
    4. Sonicate the homogenate 4 times for 10 seconds on ice each time to shear nuclear DNA.
    5. Centrifuge the homogenate at 90,000 x g or higher for 30 minutes at 4°C in a Beckman Table Top TL-100 ultracentrifuge, Beckman Airfuge or equivalent. Alternatively, clients can also centrifuge at maximum speed (15,000 – 17,000 rpm) on a benchtop microcentrifuge for 30 minutes at 4°C. The following steps should be performed as quickly as possible once the supernatant fraction is obtained.
    6. Transfer the resulting supernatant fraction to a new tube, which is kept in an ice bath, and subject it to a protein assay using a commercial Bradford assay reagent or using the standard protocol of Bradford. BSA should be used as the protein standard. The protein concentration obtained should be approximately 15-20 mg/ml or higher. If the concentration obtained is less than 1.0 mg/ml, samples should be concentrated using an Amicon Ultra-0.5 Centrifugal Filter (Millipore).
    7. Aliquot 100 µg for each lysate to be analyzed with KAM-900P and keep it on ice if it is to be used immediately.
    8. Store any remaining lysates at -70°C for subsequent validation studies. Label the microcentrifuge tubes and freeze them immediately.
  5. Additional Notes for KAM Lysate Preparation
    1. Note all cell lines are different so the suggested number of 1×106 to 2×106 cells for each sample is an estimate based on commonly used cell lines. For the validation immunoblotting service, you will need to prepare about 10 times more cells (1×107 to 2×107 cells).
    2. Cells or tissues should be processed in a timely fashion at 4°C or below if the chemical cleavage step is not used.
    3. The Kinexus Lysis Buffer with its phosphatase and protease inhibitors should be completely dissolved and kept over ice just prior to use.
    4. Protein concentration of each sample should preferably be at or above 3.0 mg/ml.
    5. 100 μg of lysate is recommended to be used, especially with the KAM-900P chip, since the phosphorylation of target proteins at specific sites is often found with very low stoichiometry. However, if sample material is difficult to obtain, as little as 25 μg of lysate has been successfully used. (Note: The same amount of protein from each sample to be analyzed together must be applied to the microarray for optimal comparison purposes).
    6. To minimize the volume and maximize the protein concentration of lysates, the lysis buffer used to recover the scraped cells from a culture dish can be transferred to the next dish if multiple dishes of cells for the same sample are to be used for lysate preparation. It is advised to use the minimal amount of lysis buffer for lysate preparation to achieve the protein concentration required for the KAM-900P antibody microarray analysis.
    7. Nuclear DNA shearing by sonication or needle passing is necessary and cannot be omitted.
    8. The highest centrifugal forces achievable on a microcentrifuge should be used to prepare the detergent-soluble fraction.
    9. Detergents should be omitted from the lysis buffer if a particulate-solubilized fraction is to be prepared and analyzed.
    10. Supernatants should be separated from pelleted precipitates and frozen as quickly as possible if the chemical cleavage is not performed. Removal of an aliquot for the protein assay is suggested so that the bulk of the lysate sample can be frozen quickly to preserve the phosphorylation state of the proteins in the extract.
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.

Preparation of Cell and Tissue Pellets

An additional charge of $200 per sample will apply for submission of cell pellets to be processed at Kinexus. A sufficient number of cells (>2 x 106 cells) should be provided for each sample to be subjected to KAM-900P analysis. If Kinetworks™ multi-immunoblotting is desired for validation of the KAM-900P results, the number of cells required is ten-fold higher (>2 x 107 cells).

A. Adherent Cells


  1. Remove the medium and rinse the cells in dish with ice-cold PBS once;
  2. Detach cells with trypsin as one does in passaging cells or scrape the cells with a rubber policeman, followed by the addition of equal volume of medium;
  3. Collect cells in a 15-ml conical tube and centrifuge at 500 x g for 2 minutes at 4•C in a swinging bucket benchtop centrifuge;
  4. Wash the pellet twice with ice-cold PBS thoroughly, (the presence of serum from medium could skew the protein assay) and remove as much PBS as possible (the presence of liquid residue dilutes the sample and may also result in the damage of cells during freezing process); and
  5. Freeze the pellets for shipping. Pellets must be shipped on dry ice.

B. Suspended Cells


Simply follow Steps 3-5 above for “A) Adherent Cells” and freeze the cell pellet immediately.

Pellets must be shipped on dry ice.

C. Tissues


An additional charge of $200 per sample will apply for submission of tissue samples to be processed at Kinexus. Freshly harvested tissues are preferred if possible. When harvesting, the tissues should be cut into small pieces on the surface. Wrap the tissues individually in tinfoil and snap freeze them in liquid nitrogen for 10 minutes before storing them at -80 •C. The tissues should be shipped on dry ice.

Storage of Samples

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 KinexTM KAM 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 KinexTM 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.

Dry Ice Shipments

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.

Shipping Details

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:

KinexTM 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.

Forms to be Completed

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.

A. Service Order Form (KAM-900P-SOF or KAM-1100-SOF)
Please ensure:
  • Address and contact name and numbers are specified
  • Billing or accounting information is completed
  • Any quotations are listed in the billing sections
  • Include a Purchase Order, Visa or MasterCard number for payment
  • The form is signed and dated

B. Service Identification Form (KAM-900P-SIF or KAM-1100-SIF)
For each sample submitted, please ensure the following:
  • At least 100 µg of protein is provided for each sample to be analyzed, 2 samples per screen
  • In Section A, the customer must assign a unique Client Screen Identification Name to correlate the proteins to be analyzed for each sample submitted
  • In Section B, the type of analysis (Kinex™ Screen Name) for each sample is specified.
  • For Section C, your sample(s) are identified by completion of Client Supplied Non-Confidential (NSDF-LY) or Confidential (CSDF-LY) Sample Description Forms. Make sure that the Client Screen ID Name in Box A of these forms, matched the Client Screen ID Name in Box A of the KAM-SIF forms
  • In Section D, the level of confidentiality is indicated for correct pricing
  • The form is certified correct and signed and dated

C. Sample Description Forms
Customers should choose which type of Sample Description Form is applicable to their lysate samples. The Non-Confidential Sample Description Form (NSDF-LY) is required to obtain the lower, non-confidential price. One form is required to be completed in full for every cell or tissue lysate submitted at this pricing level. If your samples are confidential, the the Confidential Sample Description Form (CSDF-LY) should be used.

For each lysate submitted, please ensure the following:
  • Each sample tube is labeled and properly identified on the form in Section B, including the final concentration and volume
  • A minimum of 100 µg of protein is provided for each sample submitted
  • Please be as accurate as possible in completing the Non-Confidential Form. A Technical Service Representative may contact you for additional information regarding any sample details that are unclear
  • The form is certified correct and signed and dated

D. Proteomics Services Agreement
A Proteomics Services Agreement is required to be signed before the first order can be processed. This Agreement is required to be signed and dated by an authorized representative, typically a Senior Officer, Senior Scientist, Principal Investigator, or Director of Research, before the first order can be processed, but does not have to be signed again for repeat orders. The Proteomics Service Agreement is typically valid for 15 years. If you require changes or modifications to be made to our standard service agreement, please email sales@kinexus.ca to request a Microsoft Word version of the document so your requested changes can be made directly into the agreement and emailed back to us for our final approval.


E. Courier Airway Bill
Airway bill for Federal Express or any courier that accepts dry ice shipments if the samples must be sent frozen.

Complete the airway bill and specify:
  • Priority overnight delivery
  • Bill transportation charges to your institute
  • If chemical cleavage of the samples is not performed and samples must be sent frozen, use sufficient dry ice to last several days into a large Styrofoam shipping container
  • Dry ice is a “hazardous” item, so ensure proper labels are attached to the outside of the box
  • Do not specify Saturday delivery or hold at courier location
  • Contact the courier to pick up the samples from you institute before the cut off time.
  • For shipments coming from within Canada or the United States, it is preferable to ship any day from Monday to Wednesday. Do not ship on a Thursday or Friday.
  • For international shipments coming from outside of North America, the best day to ship is on a Monday to ensure arrival in Canada for delivery later the same week
  • Customers should e-mail the date of shipment and the courier airway bill number with number of samples to Kinexus at info@kinexus.ca to ensure we can track and monitor your package in transit
  • For customers located outside of Canada, 3 copies of a commercial invoice are required to accompany your shipment (see below)

FOR U.S AND INTERNATIONAL CUSTOMER ONLY

F. Commercial Invoice (not required by Canadian customers)
Please complete one of the two attached commercial invoices (one for regular shipping and the other with dry ice) as applicable with the following information:
  • Date of exportation
  • Shipper name, address, and telephone number
  • Country of export and country of origin
  • Name of courier and the airway bill number
  • Number, type and total weight of package(s)
  • Total declared value of shipment (number of samples x $1.00 per sample) and please specify currency
  • Date, name, signature, and title of authorized person
  • Include three (3) copies of the commercial invoice on the outside of the package along with the airway bill

The regular Shipping Commercial Invoice should be used if the lysate samples are obtained from cells and tissues that have been subjected to chemical cleavage and/or homogenized in SDS-PAGE sample buffer (for immunoblotting validation studies). For lysate samples or cell/tissue pellets that must be shipped frozen, use the Shipping Commercial Invoice that corresponds to a dry ice shipment.

Please ensure 3 copies of a signed commercial invoice accompany your shipment which specifies your samples are “non-hazardous, non-infectious, and non-toxic and for research purposes only”. Since the samples are not for resale, the value of your shipment should be priced low, we recommend $1.00 per sample, to avoid paying additional duties and taxes on entry into Canada. It is also 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. If we know your package tracking number, we can often pick up your package if it misses the cut off time for the courier delivery. We will send an e-mail confirmation once your shipment arrives safely at our facility.

The international air waybill is required for all international shipments. It is your customs declaration, which can possibly be used to clear your shipment through customs at the destination. If the description on your commercial invoice is too vague or missing information, customs authorities may select the shipment for further inspection. All customs paperwork, such as the commercial invoice, must have detailed commodity descriptions. A detailed description on the air waybill and other customs documentation will help speed up the clearance time and reduce your delivery time.

KAM Antibody Microarray Reports

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. This includes calculations of Z scores, percent changes from control (%CFC), and the Kinections Pathway Mapping feature. This permits the identification of the most promising biomarkers for further validation by immunoblotting. 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-900P 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-900P chips can be ordered directly from Kinexus for follow up to experimentally validate key leads from the antibody microarray analyses.

Pricing Information

Kinexus offers the KinexTM services at different pricing levels depending on the level of confidentiality required for your samples. With the full analysis with 878 pan- and phospho-site-specific antibodies and full confidentiality, our regular price for the KinexTM KAM Antibody Microarray Services starts at US $2,998 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 40% 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. 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-900P analyses into protein phosphorylation network maps. We have produced a database of over 11,000 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$225 each, whereas quantitative maps cost US$275 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.

Follow Up Services

We highly recommend that all interesting leads generated with the Kinex™ KAM Antibody Microarray should be validated by Western blotting before proceeding to other follow-up work. Such validation is essential with any commercial or custom produced antibody microarray. To assist in this regard, Kinexus offers two cost-effective custom immunoblotting services.

Clients can use the Kinetworks™ Custom KCPS 1.0 (Multi-Antibody) Protein Screen, where any 18 antibodies used on the KAM-900P chip can be selected, and we can test whether they correctly detect their target proteins and phosphosites in your experimental model system. If there are multiple samples to test, it is often advisable to have a pre-screen performed where equal aliquots of sample lysates are pooled and then tested to confirm the antibodies are detected on a Western blot. Alternatively, with the Kinetworks™ Custom KCSS 1.0 (Multi-Sample) Protein Screen, up to 8 different samples can be probed with up to 3 different antibodies, provided the molecular masses are significantly separated by SDS-PAGE. Lysate samples for Kinetworks™ analyses may be shipped without refrigeration to Kinexus if they are boiled and stored in SDS-PAGE sample buffer. More information about these Kinetworks™ services and the necessary forms can be download from our website at http://www.kinexus.ca/ourServices/immunoblotting/custom_profiling/custom_profiling.html.

The availability of these Kinetworks™ Custom screens is another important distinguishing feature of our antibody microarray services as clients can have their research leads conveniently and cost-effectively confirmed. The cost savings arising from the use of the Kinexus discovery platform becomes immediately apparent when one considers the purchase costs of individual antibodies and the labour necessary to confirm key antibody results obtained with other antibody microarrays. In addition, once the results are confirmed by Western blotting, clients can correlate their data with thousands of other data points from hundreds of different model systems using our KiNET databases, which contain the results from thousands of previous Kinetworks™ Immunoblots or Kinex™ Antibody Microarray analyses. Over 500 scientific publications have been published that reference the Kinexus Services, of which more than 150 are directly related to the Kinex™ Antibody Microarray Services.

In addition to the Kinetworks™ Custom Immunoblotting Services to validate leads, Kinexus can assist with many other aspects of your research project from start to finish. Other services that can be used in combination with our Kinex™ Antibody Microarray services include the following:

In vivo services – send us your experimental compounds, proteins or oligonucleotides and we will perform the treatment of cells according to your specification and generate lysates for testing with our microarrays;
  • Tissue or cell pellet processing – send us your cell pellets or tissues and we will prepare lysates for you;
  • Mass spectrometry identification of antibody cross-reactive proteins;
  • Custom Graphics – we can prepare pathway charts and bar graphs for your scientific publications;
  • Custom Antibody Microarrays – we can print custom microarrays with hundreds of antibodies selected from our antibody library or supplied by you for your own internal research programs;
  • Custom Antibody Macroarrays – we can print custom nitrocellulose or glass slide arrays with 10 to 100 or more antibodies from our antibody library or provided by you;
  • Custom Reverse Phase Lysate Microarrays – we can print custom microarrays with hundreds of cell or tissue lysates to allow for further evaluation of the biological robustness of biomarkers identified through our Kinex™ Antibody Microarray services. These can be sourced from Kinexus or supplied by you;
  • Custom Lysate Macroarrays – we can print custom nitrocellulose or glass slide arrays with 10 to 100 or more cell/tissue lysates slected from our library or supplied by you; and
  • Kinase and phosphatase substrate or compound inhibitor profiling services with more than 450 active protein kinases and phosphatases to choose from.

Kinexus also offers free services and open access on-line databases to clients which include the following:

KiNET™ Antibody Microarray (KiNET-AM) DataBase (www.kinet-am.ca) – clients can directly compare their Kinex™ Antibody Microarray results with lysates from thousands of other experimental model systems analysed with the same methodology;
KiNET™ Immunoblotting (KiNET-IB) DataBase (www.kinet.ca) – clients can compare the results from their validation immunoblotting data with hundreds of other experiments from hundreds of other model systems.
PhosphoNET KnowledgeBase (www.phosphonet.ca) – clients can compare interesting phosphosites identified by our microarrays with over 180,000 confirmed and 790,000 additional predicted human phosphosites to learn about their evolutionary conservation in up to 20 different species as well as the top 50 kinases predicted to phosphorylate these sites;
KinaseNET KnowledgeBase (www.kinasenet.ca) – clients can retrieve comprehensive information on over 536 human protein kinase.
DrugKiNET KnowledgeBase (www.drugkinet.ca) – clients can identify the most potent inhibitors experimentally verified for all of the human protein kinases tracked on our microarrays as well as predicted inhibitors for off target kinases.
OncoNET KnowledgeBase (www.onconet.ca) – clients can obtain information about the expression and mutation of many of the proteins tracked on our microarrays in diverse types of human cancers.
TranscriptoNET KnowledgeBase (www.transcriptonet.ca) – clients can compare expression levels identified by our microarrays with the mRNA levels for over 20,000 human genes in 600 different human organs, tissues and cell lines.
KinATLAS (www.kinatlas.ca) - clients can identify protein-protein interactions in a cell and tissue specific manner with this pathway mapping site that also tracks kinase-drug interactions.
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