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Kinetworks™ Multi-Immunoblotting Services

Introduction

The Kinetworks™ signal transduction protein profiling services are a convenient, reliable and cost-effective solution to assist scientists in the discovery of productive research leads. These services utilize a proprietary technology based on multi-immunoblotting that generates a unique identification pattern for each sample analyzed and can provide information about the quantitative expression level for each protein detected, its covalent modification, its subcellular location and the identification of protein-protein interactions. It is highly accurate, since the detection of a target protein is based on its immunoreactivity and apparent molecular mass. Kinexus has undertaken the testing of more than 6000 commercial antibodies to select the most potent and specific antibodies for detecting low abundance proteins over a wide range of model systems. The Kinetworks™ approach, which has been under development and field-tested for over eight years, is faster and more sensitive for specific protein detection and offers greater versatility and reproducibility than many other proteomics methods. Presently, with over 875 antibodies Kinexus can track more than 700 unique cell signalling proteins and phosphosites and several hundred unknown cross-reactive proteins. Only our Kinex™ antibody microarray services provide a cheaper alternative to profiling changes in protein expression and phosphorylation than our Kinetworks™ protein profiling, but the microarray approach is less accurate and generates a high degree of false positives and false negatives. Our Kinetworks™ Custom Antibody Screen (KCPS 1.0) is the recommended route to validate interesting results that are generated from our Kinex™ microarray services.

Kinexus currently offers 4 different standard analytical signal transduction protein phosphosite profiling services. In addition to our standard screens, we offer 2 flexible custom screening services including the KinetworksTM Custom (Multi-Antibody) Protein Screen (KCPS 1.0) that allows clients to choose any 18 antibodies of interest out of more than 650, which are available from Kinexus. The KinetworksTM Custom (Multi-Sample) Screen (KCSS 1.0) allows clients to choose up to 3 target proteins (of diverse molecular weight) quantitated in 8 different samples side by side on the same immunoblot. The Kinex™ antibody microarray service KAM-880 utilizes 877 antibodies and tracks over 500 distinct signalling proteins in duplicate in two samples on the same microarray slide. For more information about this service, please review the KinexTM Customer Information Package available on-line from our website at www.kinexus.ca. Clients can compare and correlate their experimental results with our Kinetworks™ services with thousands of other Kinetworks™ analyses that Kinexus has performed over the last 17 years by freely querying our KiNET databank on-line (www.kinexus.ca/kinet).

Our Kinetworks™ analyses of over 10,000 cell/tissue samples have revealed that there is a high degree of variability in the expression and phosphorylation levels of signal transduction proteins in diverse cell and tissue type that is species, organ, tissue and gender dependent. Consequently, we recommend that our standard KinetworksTM Screens be performed initially to ascertain which target proteins are detectable in your experimental model systems before conducting any Custom Screens. All experimental results should be reproduced prior to publication in accordance with good laboratory practices.

Kinexus provides both qualitative and semi-quantitative analyses of the expression and phosphorylation states of protein kinases and cell signalling proteins in cell and tissue samples as part of the KinetworksTM screening service. The qualitative analyses include TIFF files of the immunoblots that feature the detected target signalling proteins (see example of a Kinetworks™ immunoblot image below). The KinetworksTM analysis has been specially optimized to reveal band shifts in signalling proteins on SDS-PAGE gels that may arise from their phosphorylation. The quantitative analysis of the strength of the enhanced chemiluminescence signal for each target protein is provided in a Microsoft Excel spreadsheet. For multiple samples within the same profiles, Kinexus provides Comparison Reports for the target proteins and graphs the data against the control samples. The KinetworksTM screening service is unmatched for the information that it provides about multiple kinase expressions and phosphorylations in a single assay. All the KinetworksTM Screens have been optimized to perform in human, mouse and rat model systems, but can also work for many protein targets in cow, pig, dog, rabbit, chicken, frog, starfish and other various model systems. You can contact a Customer Service Representative at info@kinexus.ca if you would like to view an example of what our KinetworksTM Screens may look like in your particular model system.

Quantity of Lysate Required

Recognizing the importance of elucidating the inhibition profiles of compounds, Kinexus has developed an innovative microarray-based profiling platform for screening small-molecule protein kinase inhibitors. The cores of this technology consist of a protein kinase micorarray and a biotinylated proprietary ATP probe developed at Kinexus. The probe has been shown to display affinity for the majority of protein kinases tested by our internal research work. Co-incubating a test compound with the biotinylated ATP probe on the protein kinase microarray allows simultaneous determination of the affinity of the compound against hundreds of protein kinases on the array on a competition binding basis. The kinases to which the compound exhibits binding will experience a reduction of the binding of the ATP probe, and the remaining ATP probe covalently bound to the kinases on the array can be detected with the fluorescently-labeled streptavidin conjugates. Compared to the aforementioned Kinomescan™ and Kinativ™, advantages of our microarray-based inhibitor profiling platform include higher throughput, small amounts of compound required, and the use of active protein kinases expressed in eukaryotes. Without the requirements for any sophisticated analytical instrument such as quantitative PCRs and mass spectometers, our technology presents a quick and effective approach to survey a broad spectrum of the human kinome for compound inhibition profiling prior to any in-depth assays to be perfomed. Thus, the protein kinase microarray-based inhibitor profiling complements well with our existing Kinase Inhibitor Profiling services that are based on measurement of phosphotransferase activity. Any hits from the Kinex™ Kinase Microarray can be confirmed with our Kinase Inhibitor Profiling services against the very same preparations of protein kinases.
  • 20 mM MOPS, pH 7.0 (any other buffer 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% 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 phenylmethylsulfonylfluoride (to inhibit proteases);
  • 3 mM benzamidine (to inhibit proteases);
  • 5 µM pepstatin A (to inhibit proteases);
  • 10 µM leupeptin (to inhibit proteases);
  • 1 mM dithiothreitol (to reduce disulphide linkages)
The final pH of the homogenizing buffer should be adjusted to 7.2. Please note that Kinexus is willing to send an aliquot of our lysis buffer for a fee to any customer who provides 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 (components 9-12). Clients must add their own 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. Important things to remember are that the cells or tissues should be processed quickly at 4°C or less. Homogenization should not be performed in too large a volume to obtain lysates at the concentration required. The detergent-soluble fraction should be obtained as quickly as possible after the cells or tissues are homogenized. Sonication is required and cannot be omitted. The highest centrifugal forces available should be used to generate the detergent-soluble fraction. The supernatants should be frozen as quickly as possible if a protein assay cannot be performed immediately.

Preparation of Cell Lysates

A. Adherent Cells


Remove medium from culture dishes containing about 1×107 to 2 ×107 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), or 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 is a crucial step and cannot be omitted;

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

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) 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 (BSA) should be used as the protein standard. Make sure that the protein concentration is determined before the addition of SDS-PAGE Sample Buffer.

B. Suspension Cells


Place medium containing cells in appropriate sized tube and spin 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 200 μl ice-cold lysis buffer per 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 more for 30 min at 4°C in a Beckman Table Top TL-100 ultracentrifuge or Beckman Airfuge;

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) 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 (BSA) should be used as the protein standard. Make sure that the protein concentration is determined before the addition of SDS-PAGE Sample Buffer.

Preparation of Cell Pellets

For an additional fee of $200 per sample, Kinexus will process your cell pellets into a lysate for processing with any of our KinetworksTM screens. To prepare your cell pellets for shipping to Kinexus, please follow steps 1-4 below and label the tubes containing your pellets accordingly. Cell pellets must be shipped on dry ice. Clients may need to prepare as much as 2 x 107 cells to ensure sufficient quantity.

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, 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); Remove as much PBS as possible (The presence of liquid residue dilutes the sample and may also result in the damage of cells during the freezing process);
  5. Freeze the pellet for shipping. Pellet must be shipped on dry ice.

B. Suspension Cells

Simply follow steps 1-3 in the Section 3B and freeze the cell pellet immediately. Pellets must be shipped on dry ice at the expense of the client.

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.

Sample Buffer Preparation

We recommend the final composition of SDS-PAGE Sample Buffer in the sample be: 31.25 mM Tris-HCl (pH 6.8), 1% SDS (w/v), 12.5% glycerol (v/v), 0.02% bromophenol blue (w/v), and 1.25 % beta-mercaptoethanol. The cell/tissue samples should be boiled for four (4) min at 100°C in the SDS-PAGE Sample Buffer. (See Appendix A for detailed instructions on preparing the Sample Buffer). Please note that Kinexus is willing to send customers an aliquot of Sample Buffer (but without the beta-mercaptoethanol) for a small fee. If this option is of interest, please contact a customer services representative for details.

Preparation for Storage and Shipping of Samples

The final protein concentration of the cell/tissue samples should be 1 mg/ml in SDS-PAGE Sample Buffer as specified by Laemmli (Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-684). For all screens, the minimum acceptable protein concentration of the cell/tissue samples in the SDS-PAGE Sample Buffer is 0.6 mg/ml and the maximum concentration is 2.0 mg/ml. Please record the actual concentration and volume of each sample on the Sample Description Form (Box B of KW-NSDF-01 or KW-CSDF-01).

For all KPSS standard screens, exactly 500 µg (i.e. 500 µl of 1 mg/ml protein) of boiled cell/tissue extract protein in the SDS-PAGE Sample Buffer should be aliquoted into a 1.5-ml Eppendorf screw cap vial. For the Custom Screens, the KCPS-1.0 Multi-Antibody Screen also requires at least 500 µg of cell/tissue lysate, while the KCSS-1.0 Multi-Sample Screen requires at least 50 µg per sample. There should be one vial per sample for each screen requested, except for the KCSS-1.0 Multi-Sample Custom Screen which can have up to 8 different samples. The vials should be clearly labeled with an indelible marker with an unique identification number (recorded on the Sample Description Form), parafilmed, and then put into a secondary support container such as a 50-ml conical centrifuge tube to provide extra protection to prevent accidental leakage during shipping. It is not necessary to refrigerate or freeze the samples during shipping once they are in SDS-PAGE Sample Buffer.

Pricing Information

Kinexus offers the KinetworksTM services at two different pricing levels depending on the level of confidentiality required for your samples. Our regular prices for any of our Standard Services start at US $1,498/sample for each sample submitted for analysis if the sample information is to remain fully confidential.

At this pricing level, only the species needs to be disclosed. To receive a 50% discount off the regular price, or pay $749/sample for each standard screen ordered, Kinexus requires the Client Supplied Non-Confidential Sample Description Form (KW-NSDF-01) be completed in full (Sections A-K) including species, organ, tissue, cell, cell state, fractionation, perturbation, and treatment for each sample being analyzed. The cost of our Custom Services is also dependant on the amount of information that can be disclosed about your samples. At the non-confidential pricing level, the cost of our Custom Services is US $649 to $880 per sample, while confidential pricing is US $1,098 to $1,498 per sample. For volume discounts or quotations for large orders, please contact the Director of Sales & Marketing at 1-866-KINEXUS (option 2 on the telephone directory) or email sales@kinexus.ca.

Follow Up Services

Kinexus offers several auxiliary services to complement your KinetworksTM data including a drawing service to obtain presentation-ready Microsoft PowerPoint slides or publication-ready figures (e.g. colored overlays of immunoblot images or bar graphs of the quantified results of Kinetworks™ analyses). The differential regulation of detected proteins can be visualized in various colour combinations as presented below. Clients can also visit the Kinexus website and use the tables that list the target proteins to Internet sites with more detailed information about these proteins. 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 free KiNET on-line database. For more information about any of these services, please contact one of our customer service representatives at info@kinexus.ca.
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