KINEX™ Reverse Lysate Microarrays

The vast majority of the proteins encoded by the genomes of humans and other species remain poorly characterized with respect to their distribution, functions and regulation. Our Standard and Custom Kinex™ Reverse Lysate Microarray (KRLM) services uniquely permit the rapid simultaneous quantification of specific proteins for their distribution and regulation from analysis of hundreds of lysates from diverse cells and tissues. With this convenient, cost effective and powerful service, clients can have about 250 cell and tissue lysates probed by Kinexus with one of hundreds of antibodies of their choice at a time. Our Standard service with cell and tissue lysates prepared in-house at Kinexus permits the characterization of cell signalling proteins for their regulation in response to a wide range of drugs and hormones in 12 diverse human tissue cell lines, in maturing frog oocytes, and their distribution in monkey, mouse and rat tissues. On our Standard Kinex™ Reverse Lysate Microarray, for the drug and hormonal treatments, the tumour cell lysates have been generated from dose response and time course studies that have been performed by Kinexus. Our Custom service utilizes cell and tissue lysates that are provided by our clients. These lysates are printed in triplicate at four dilutions on the Kinex™ Reverse Lysate Microarrays to ensure linearity of detection when the chip is probed with an antibody that specifically recognizes the target protein. Deliverables include a TIFF image of the scanned antibody probed microarray and an Excel spreadsheet with quantification of all of the lysate spots on the microarray. Clients may choose the probing antibody for use with the Kinex™ Reverse Microarray from our panel of over 800 available antibodies or have Kinexus perform the analysis with antibodies that they supply. This service is particularly ideal for the characterization of novel proteins that are identified initially with our Kinex™ Antibody Microarray service and have been subsequently shown with our Kinetworks™ Custom services to be specific for immune detection.

As the specificity of immunoreactivity of antibody can vary depending on the cell or tissue extract under examination, it is highly recommended to confirm key results by immunoblotting. Any interesting Kinex™ results that clients may wish to follow up can be validated by Western blotting with the same lysates used on the Standard Kinex™ Reverse Lysate Microarray. Our Custom Kinetworks™ KCSS 1.0 service allows clients to choose any 3 target proteins (of different molecular weight) to be quantified in 8 different samples side-by-side on the same immunoblot. The cell and tissue lysate samples from our In Vivo Services, which are used on the Kinex™ Reverse Lysate Microarray, may be used at no extra cost with the Custom Kinetworks™ KCSS 1.0 service. The availability of Kinetworks™ analyses is an important distinguishing feature of our Kinex™ microarray services as clients can have their research leads conveniently and inexpensively confirmed. Further information about the expression or phosphorylation of leads can be obtained through query of our free KiNETTM on-line DataBank with results from over 6000 Kinetworks™ immunoblots.

A notable advantage of the Kinex™ Reverse Lysate Microarray approach as compared to studies with tissue microarrays is the better retention of soluble cytoplasmic proteins in the reverse lysate microarrays. Kinexus has determined that about three quarters of most cell signalling proteins are found in the cytosol of cells, which is typically lost in fixed cell preparations or stain in a very diffuse pattern and are difficult to quantify for expression. Consequently, changes in protein expression or phosphorylation in response to a cellular perturbation are more accurately represented with the Kinex™ Reverse Lysate Microarray.
The following is a brief listing of the lysates from human tumour cell lines and tissues from other species that are currently available on the Kinex™ Reverse Lysate Microarray. Further details are provided in the Appendices A and B. The precise content on the Kinex™ Reverse Lysate Microarray is subject to change as we continually aim to improve and expand the range of cell and tissue lysates that it features.

With our Custom Kinex™ Reverse Lysate Microarray service, clients can have microarrays printed that feature 250 or more lysates that they have provided. This could be extremely useful for analysis of large sets of clinical samples for promising biomarkers or for evaluating the effects of a wide range of drugs or hormones on cell lines. Due to the high set up costs with printing custom microarrays, clients must order a minimum of 20 slides for the Custom Kinex™ Reverse Lysate Microarray service. Clients should complete the Client-Supplied Lysate Description Form (KRLM-SDF-LY) provided in this client information package. If you do not wish to provide the requested information, then full confidential pricing charges will be applied. Note that due to the large number of lysates to be described, we request that clients provide the sample descriptions in an electronic format in either a Microsoft Word or Excel document, and as a printed copy with the completed KRLM-SDF-LY form. We request that the lysates are shipped frozen on dry ice to Kinexus at a concentration of approximately 5 mg/ml with a minimum of 40 µl volume.

With our Standard Kinex™ Reverse Lysate Microarray service, all of the cell lysates and a wide selection of possible probing antibodies are available as part of this very convenient and cost effective service. A complete listing of the antibodies available from Kinexus for probing the KRLM chips is available in MS-Excel format for downloading from the following URL on the Kinexus website: http://www.kinexus.ca/ourServices/microarrays/antibody_microarrays/antibody_microarrays.html. These antibodies are used in the creation of Kinex™ Antibody Microarray. We also provide the option for our clients to submit their own antibodies for use in our Standard and Custom Reverse Lysate Microarray Services in addition to those offered by Kinexus. In this event, clients should complete either the Client-Supplied Non-Confidential Antibody Description (NSDF-AB) or the Client-Supplied Confidential Antibody Description (CSDF-AB) forms provided in our customer information package for these services. If you do not wish to provide the requested information on the NSDF-AB form, then full confidential pricing charges will be applied. We recognize that not all of the requested information about commercial antibodies may be available to our clients, so please provide as much information as you are able to qualify for Non-Confidential pricing.

For antibodies provided by the client, we would need the antibody as concentrated as possible, preferably at around 1 mg/ml. We require approximately 30 µg of most pan-specific antibodies, and about 10 µg of most phosphosite antibodies following their affinity purification. As there are large variations in the potency and specificity of antibodies, and every antibody is unique, and we may be able to perform the Kinex™ Reverse Lysate Microarray service with less antibody if it still works well at very high dilution by Western blotting.

We ask that the client supplied cell and tissue lysates are provided at a concentration of ~5 mg/ml with a volume of at least 40 µl in small Eppendorf vials. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the Client-Supplied Lysate Description Form (KRLM-SDF-LY)), parafilmed, and then put into another support structure such as a styrofoam rack or plastic box that holds vials to provide extra protection during shipping. These lysates should be shipped frozen with plenty of dry ice in case the samples are held up at Canadian Customs. We have provided a commercial invoice statement for shipping cell lysates in our client information package for these services.

We recommend that the client supplied antibodies are provided as concentrated as possible and within screw cap vials. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the Client-Supplied Antibody Description forms (NSDF-AB or CSDF-AB)), parafilmed, and then put into another support structure such as a 50-ml conical or centrifuge tube to provide extra protection during shipping. It is not critical to ship the antibody preparation in a frozen state. We have provided two possible commercial invoice statements with this client information package in the event that you may wish to ship your antibodies frozen on dry ice instead.