Immunohistochemical characterisation and PEPSCAN ANALYSIS of monoclonal antibodies against D-type Cyclin-dependent kinase Cdk6
| Autoři | |
|---|---|
| Rok publikování | 2000 |
| Druh | Článek ve sborníku |
| Konference | Cells II |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Genetika a molekulární biologie |
| Klíčová slova | Cyclin dependent kinase Cdk6 |
| Popis | The complexity of regulation of protein Rb interacting with Cdk6, cyclinD and Cdk inhibitors represents a critical checkpoint in cellular signal transduction triggered by mitogenic or antiproliferative stimuli. Such a highly dynamic qurternary complex represents a functional unit that is not only involved in major decisions on the fate of a cell, but is also commonly deregulated in certain types of cancer. Cdk6 protein could be an appropriate marker of proliferation. The aim of the study was to prepare a panel of monoclonal antibodies distinguishing different epitopes of Cdk-6 (cyclin- dependent kinase 6) protein. Using hybridoma technology, dot blot, immunoblotting, PEPSCAN ELISA test and immunohistochemical staining, we characterized a panel of monoclonal antibodies against Cdk6 protein. 32 types of hybridoma cells were prepared and subsequently tested for production of monoclonal antibodies. 24 hybridomas were selected and further analyzed for reaction with 21 different target peptides using PEPSCAN ELISA test. Finally, 20 hybridoma cells, showing reaction against individual peptides, were subjected to immunoblotting in order to confirm interaction with Baculo Cdk6 (+), Baculo Cdk6 (-) and Bacter Cdk6 (+) proteins. We also used immunohistochemical staining of tissues using 6 monoclonal antibodies showing different reaction with Baculo protein Cdk6 (+) as well as with target peptides. High correlation was found between the recognition of a target antigen and the immunolocalization (nuclear, cytoplasmic) staining. We have detected Cdk6 positive cells in the proliferative compartment of the following normal human tissues: intestinal epithelium, placenta of 6-12 weeks old fetuses and activated lymphatic tissue. We selected 4 monoclonal antibodies specifically recognizing particular epitopes of Cdk-6 protein that were proved to be a useful tool for immunochemical analysis as well as immunohistochemistry. |
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