engleski

Isolation and characterization of sequence specific polyclonal antibodies raised in rabbits against C-terminus of insuline receptor betha-subunit kinase domain

Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose levels, it is secreted by the beta cells of the pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. The insulin receptor (IR) is a transmembrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its downstream substrates become tyrosine phosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response like insertation of glucose transporter 4 (GLUT4) in to the plasma membrane. One important mechanism by which insulin signaling is regulated involves the protein tyrosine phosphatases (PTPs), which may either act on the insulin receptor itself and/or its substrates. In our work we have isolated and purified antibodies for the detection of betha-subunit of IRKD. Antibodies are used to confirm the expression of IRKD recombinant protein using Baculovirus expression system in Sf9 cells. Two peptides have been synthesized whose amino acid sequences are equal to the different sequences of the C-terminal end of the  -subunit of the IRKD. The peptides have been used as antigens for immunization of rabbits. During the immunization time of 6 months rabbits have produced sequence specific polyclonal antibodies. Antibody production in the rabbit serum has been detected by ELISA test. Produced antibodies have been isolated from the rabbit’ s immune serum and purified by following methods: precipitation by ammonium sulfate and affinity chromatography on protein A and petide-sepharose columns. The specificity of produced antibodies were determinated by Elisa-test and Western-blot analysis.

inzulinski receptor; sinteza peptida; imunizacija; protutijela; Bakulovirusni ekspresijski sustav; ELISA-test; afinitetna kromatografija; Western-blot analiza

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