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Kinex™ Antibody Microarray Services

Introduction

The Kinex™ signal transduction protein profiling services are a convenient and very cost-effective solution to assist scientists in the broad discovery of productive research leads such as biomarkers. These services utilize our unique antibody microarrays to track the differential binding of dye-labeled proteins in lysates prepared from cells and tissues. The results can provide productive insights into differences in protein expression, phosphorylation and protein-protein interactions, and define antibody reagents that can be used to follow up these findings. However, as non-denatured proteins are analyzed by this method, there is increased opportunity for false positives and false negatives due to antibody cross-reactivity and blocked epitopes in protein complexes. To partly offset this problem, we also offer a chemical cleavage step to produce large peptides that retain most phosphorylation sites. Even with this additional step, we highly recommend that any interesting Kinex™ results that clients wish to follow up should be first validated by Western blotting, for example with our Kinetworks™ multi-immunoblotting services. Our Custom Kinetworks™ KCPS 1.0 service permits up to 18 antibodies from our Kinex™ antibody microarrays to be used at a time for validation studies by Western blotting. The Custom Kinetworks™ KCSS 1.0 service allows clients to choose any 3 target proteins (of different molecular masses) to be quantified in 8 different samples side-by-side on the same immunoblot. The availability of Kinetworks™ analyses is an important distinguishing feature of our antibody microarray services as clients can have their research leads conveniently and cost effectively confirmed. Further information about the expression or phosphorylation of leads can be obtained through query of our KiNET™ databank with results from over 6000 Kinetworks™ immunoblots.

In our internal studies with cells from different species, only between 30 to 45% of the protein changes detected on the Kinex™ KAM antibody microarrays were reproduced by immunoblotting, if no chemical cleavage step was performed. It should be appreciated that the high rate of false positives associated with our antibody microarray is an inherent problem with all other commercial antibody microarrays due to the reliance on non-denaturing conditions for immune capture of target proteins. About 20 to 25% of the Kinex™ antibody microarray-detected 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. The Kinex™ KAM-880 chip has typically 25-times the antibody coverage, it uses 5-10-times less cell/tissue lysate protein, and it yields duplicate measurements at 10-30-times less cost than a Kinetworks™ immunoblot analysis. Therefore, the antibody microarray is a particularly attractive initial route for taking a system biology, proteomics approach to studying human disease or an experimental model system.

Kinexus has independently tested over 6000 different antibodies to identify the best immunological reagents to track popular cell signalling proteins. The top 20% of these antibodies, which have been proven in-house by Kinexus to perform well in Western blotting applications, were incorporated into our latest generation Kinex™ KAM microarray. Our current Full Kinex™ Service using the KAM-880 chip with two samples analyzed at a time utilizes ~518 pan-specific antibodies (for protein expression) and ~359 phosphosite-specific antibodies (for phosphorylation) in duplicate for at least 333 different phosphosites, 234 different protein kinases, 44 protein phosphatases and 209 different regulatory subunits of these enzymes and other cell signalling proteins that regulate cell proliferation, stress and apoptosis; the complete list of unique target proteins and phosphosites tracked in the Kinex™ KAM-880 Antibody Microarray is available for download from the Kinexus website in an MS-Excel spreadsheet. With a 33% discounted price compared to the full analysis with 877 antibodies, it is also possible for clients to choose to have their cell and tissue samples analyzed with the KAM-880 Antibody Microarray and get back results for only the ~518 pan-specific antibodies or the ~359 phosphosite-specific antibodies. Further discounts are available if clients select our non-confidential pricing options.

With respect to the performance of the Kinex™ antibody microarrays, we have analyzed over 4000 Kinex™ KAM chips to date. The antibodies used in the Kinex™ 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 other diverse model systems. With the latest KAM-850 chip, we found 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 is ±12%). The average of duplicate measurements of the same sample on different chips had a median error range of ~8%. The dynamic range between the highest and lowest reproducible dye-bound protein signals from these Kinex™ chips was over 5000-fold. This performance exceeded that of antibody microarrays from three other competitors tested in our hands. Moreover, we have determined that the costs of using our Kinex™ service can be 20% to 50% less than the cost of purchasing competitor antibody microarrays and a researcher performing this kind of analysis in their own lab (note that the added costs of the chip scanners and quantification software license are not included in these comparisons).

One of the key differences between the Kinex™ antibody microarray chips and competitor microarrays is that we label the control and treatment cell/tissue lysate samples with the same dye, and we analyze both samples separately, but on the same chip. In our experience, the use of two dye, competitive binding systems 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 high rate of false leads. Unlike oligonucleotides such as DNA, 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. Therefore, colour changes seen with spots evident on a DIGE 2D gel may not be related to differences in protein expression at all 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 1000-fold lower than structural proteins and metabolic pathway enzymes. Consequently, these low abundance proteins are usually not evident on 2D gels without some special pre-enrichment. This is why we feel that antibody-based detection of proteins with our Kinex™ antibody microarrays or Kinetworks™ multi-immunoblots are complementary and superior methods to undertake broad studies of proteins for signalling network analyses.

As part of the full Kinex™ KAM-880FN, KAM-880FC and KAM-880FP antibody microarray services with 877 antibodies, Kinexus provides both qualitative and semi-quantitative analyses of the expression and phosphorylation states of cell signalling proteins in cell and tissue samples as determined with the KAM-877 chip. Quantification of the signal strength on each antibody spot uses a combination of both the size of the spots as well as their density of the signals. The qualitative analysis may include TIFF and JPEG files of the scanned Kinex™ antibody microarray that features the detected target signalling proteins in control and experimental samples. The quantitative analysis of the strength of the fluorescence signals for each target protein is provided in duplicate in a Microsoft Excel spreadsheet and includes the (average) percent change from the control sample, the percent range in error, Z scores, and fold-changes ratios. To view example images or a sample of a Kinex™ Report, including a Kinections Map analysis, please contact a Customer Service Representative at info@kinexus.ca.

The partial and discounted Kinex™ KAM-880EN, KAM-880PN, KAM-880EC, KAM-880PC, KAM-880EP and KAM-880PP antibody microarrays services provide results with either ~518 pan-specific antibodies or ~359 phosphosite-specific antibodies. With these antibody microarray services Kinexus provides a complete quantitative analysis of the strength of the fluorescence signals for each target protein or phosphoprotein in duplicate on the microarrays in a Microsoft Excel spreadsheet, which includes the (average) percent change from the control sample, the percent range in error, and fold-changes ratios.

The combination of Kinex™ antibody microarray analysis followed by validation studies by Kinetworks™ multi-immunoblotting is a very economical and efficient strategy for biomarker discovery. A cost analysis is available for the use of the Kinex™ KAM-880 Antibody Microarray to screen extracts from a healthy control and a diseased tissue for differences in signal transduction protein expression and phosphorylation compared to using our Kinetworks™ immunoblotting services. The price was estimated to be just over US $58,000 with non-confidential pricing using our immunobotting service only. This is actually quite cost-effective, because if a researcher had to purchase the antibodies to perform such a study in their own laboratory, it would cost upwards of US $150,000 if he or she knew even which antibodies to procure. We might expect that around 90 leads might be generated if 15% of the tested proteins underwent disease-related changes. This rate is not uncommon in our experience. However, if the Kinex™ antibody microarray analysis was deployed first and the top 90 changes were then confirmed by follow-up Kinetworks™ custom immunoblotting, the total cost for the combined services would be around US $8,300. There would be false negatives by this approach. However, this would yield about 36 highly validated leads as well as knowledge about the nature of the antibodies that could successfully detect these proteins with both antibody microarray and immunoblotting methods in follow-up studies. We are excited by the prospect that the highly integrated platform of novel proteomics services offered by Kinexus will permit our clients to significantly improve their efficiency of biomarker detection to accelerate the promise of personalized medicine.

A large body of information and instruction is provided with our KABM Services Customer Information Package. Your careful review of this package will ensure that we can offer the highest level of quality in providing our unique proteomics services to you. We have requested a lot of information from you regarding the preparation of your cell/tissue lysate samples so that we can share this in the future in our KiNET-AM database. For these rights, we have discounted our standard charges by 41% with our Non-Confidential Pricing option. You should find that the proper entry of information into the various forms provided by Kinexus will also be useful for your own reference at a later date when you receive your Kinex™ results. The KiNET-AM database is an unique open-access online resource that allows our clients to compare their Kinex™ Antibody Microarray results with thousands of other cell and tissue lysates. This is invaluable for establishing the potency and specificity of potential biomarkers.

Quantity of Lysate Required

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.
  • 20 mM MOPS, pH 7.0 (any other buffer without Tris at this pH could be substituted);
  • 2 mM EGTA (to bind calcium);
  • 5 mM EDTA (to bind magnesium and manganese);
  • 30 mM sodium fluoride (to inhibit protein-serine phosphatases);
  • 60 mM β-glycerophosphate, pH 7.2 (to inhibit protein-serine phosphatases);
  • 20 mM sodium pyrophosphate (to inhibit protein-serine phosphatases);
  • 1 mM sodium orthovanadate (to inhibit protein-tyrosine phosphatases);
  • 1 mM phenylmethylsulfonylfluoride (to inhibit proteases);
  • 3 mM benzamidine (to inhibit proteases);
  • 5 µM pepstatin A (to inhibit proteases);
  • 10 µM leupeptin (to inhibit proteases);
  • 1% Triton X-100 (can be substituted with 1% Nonidet P-40)
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 1-7, including phosphatase inhibitors (components 4-7) but no protease inhibitors and dithiothreitol (components 9-13). 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 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.

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: At this time, we do not recommend that you send samples from immunoprecipitation or antibody affinity pull-down experiments for the KinexTM Antibody Microarray (KABM) services.

Important points 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.

Cell Lysate Preparation

A. Adherent Cell Lysates


Remove medium from culture dishes containing about 1-2 million cells;

Rinse the cells twice with ice-cold PBS to remove medium residue (serum must be completely removed from cells); remove as much PBS as possible after the last rinse;

Add 200 μl ice-cold lysis buffer to 150 mm culture dish per sample (more lysis buffer can be added if cells are concentrated); (add 100 μl ice-cold lysis buffer to 100 mm culture dish)

Scrape the cells in lysis buffer, collect the cell suspension from the dishes and transfer it into a 1.5-ml microcentrifuge tube;

Sonicate four times for 10 seconds each time with 10-15 second intervals on ice to rupture the cells and to shear nuclear DNA; this step is crucial and cannot be omitted;

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 1.5-ml microcentrifuge tube;

Assay sample for protein concentration using a commercial Bradford assay reagent (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.

B. Suspended Cell Lysates


Place medium containing cells in appropriate sized tube 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;

Wash the pellet by gently resuspending the cells in ice-cold PBS, followed by centrifugation as above. Repeat once to ensure complete removal of serum;
Remove as much PBS as possible after the last wash;

Add an adequate amount of ice-cold lysis buffer to the sample (more lysis buffer can be added if the number of cells is high);

Sonicate four times for 10 seconds each time with 10-15 second intervals on ice to rupture the cells and to shear nuclear DNA; this step is crucial and cannot be omitted;

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 1.5-ml microcentrifuge tube;

Assay sample for protein concentration 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 quantification 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.

Preparation of Cell Pellets

An additional charge of $200 per sample will apply for submission of cell pellets to be processed at Kinexus. Please submit a sufficient number of cells ( >2 million cells) for processing.

A. Adherent Cells


Remove the medium and rinse the cells in dish with ice-cold PBS once;

Detach cells with trypsin as one does in passaging cells, followed by the addition of equal volume of medium;

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;

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);

Freeze the pellet for shipping. Pellet must be shipped on dry ice.

B. Suspended Cells


Simply follow steps 3-5 in Section 4A for “for adherent cells” and freeze the cell pellet immediately.

Pellet must be shipped on dry ice.

Tissue Preparation

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);

Sonicate 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.

Preparation for Storage and Shipping of Samples

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 (NSDF-LY or CSDF-LY). Kinexus requests 100 μg of cell or tissue lysate for each sample submitted for analysis with the KinexTM Antibody Microarray. 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 samples 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 and/or inadequate completion of shipping information.

Shipping Information

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. The sample vials should be sent to the address listed below by any express courier that accepts dry ice shipments. 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:

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


Please ensure 3 copies of a signed commercial invoice accompany your shipment which specifies your samples are non hazardous and non infectious. Since the 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. For shipping from outside of North America, we highly recommend send out on a Monday or Tuesday. We will send a confirmation e-mail once your shipment arrives at our facility.

Pricing Information

Kinexus offers the Kinex™ services at different pricing levels depending on the level of confidentiality required for your samples and the whether a partial or complete analyses is requested. With the full analysis with 877 pan- and phosphosite-specific antibodies and full confidentiality, our regular price for the Kinex™ Antibody Microarray Services start at US $2,998 per slide for each pair of samples submitted. At this pricing level, only the species needs to be disclosed. It is also possible to obtain a 33% discount at $1770 per slide with full confidentiality if the data is desired for only 518 pan-specific antibodies or 359 phosphosite-specific antibodies. To receive a further 41% discount off these prices, Kinexus requires the Client-Supplied Non-Confidential Sample Description Form (NSDF-LY) to be completed in full (Sections A-K) including species, organ, tissue, cell, cell state, fractionation, perturbation, and treatment for each sample being analyzed. For performance of the chemical cleavage of proteins in cell/tissue lysates, additional charges of $150 per microarray slide apply.

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.

Forms to be Completed

All of the forms necessary to use the Kinex™ Antibody Microarray (KABM) services are provided in the Appendices section of our Customer Information Package for these services. Fillable MS-Word versions of these forms are directly downloadable from the Kinexus website at http://www.kinexus.ca/ourServices/microarrays/microarrays/antibody_microarrays.html and by request by e-mail or by phone. Please contact our Technical Service Representatives by e-mail at info@kinexus.ca or by phone at 604-323-2547 Ext. 1 for all enquiries related to technical/research issues, work orders, service fees or request of fillable order forms

Follow Up Services

Kinexus offers two types of Western blotting follow-up services to cost-effectively validate the results from your Kinex™ antibody microarray. Clients can choose from the Kinetworks™ Custom KCPS 1.0 (Multi-Antibody) Protein Screen where any 18 antibodies can be selected and we will optimize it to your model system, or with the Kinetworks™ Custom KCSS 1.0 (Multi-Sample) Protein Screen send up to 8 different samples and choose up to 3 different antibodies (provided the molecular weights are significantly separated by SDS-PAGE). Once the results are confirmed by Western blotting, clients can correlate their data with hundreds of other data points from hundreds of different model systems using our KiNET database. For large numbers of samples, clients may use our Kinex™ Reverse Lysate Microarray (KRLM) services. For more information about these services, please contact one of our customer service representatives at info@kinexus.ca or visit our website at www.kinexus.ca.
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