Tyrosine Kinases essays

Tyrosine Kinases essays In this experiment we will determine whether or not epidermal growth factors (EGF) stimulate target cells, through the receptor tyrosine kinase (RTK) (3). RTKs are transmembrane proteins that receive an extra cellular message, and transform it into an intracellular message. When inactive the RTKs reside as monomers in the lipid bylayer of the cell, until they are dimerized (2). At the same time of this dimerization the RTK then binds its ligand, EGF is a possible ligand. This action causes a conformational change that allows the RTK to be autophophorilated (1). Phosphorylation can happen on a few different side chains of the RTK. Such as serine, threonine, and tyrosine. We are concerned with the phosphorylation that occurs on the tyrosine. Phosphorylation of these side chains occurs at the exposed hydroxyl end of the amino acid. A hydrolysis reaction occurs and each dimmer yields phosphate groups to each other to allow the autophophorylation event (4). By knowing that the tyrosine side chains are phosphorylated we can perform a western blot and then using a specific antibody we determine if our EGF has caused the RTK to be phosphorylated (3). To start we took serum starved COS-7 cells that were ruffly 100 percent confluent, and were separated into a six well plate, and added to five wells different media. Before this we removed the existing media from the cells. In separate wells we added 2mls of acetyl choline 1ml/mg, EGF 2Â µl/mg, 10% FCS, serum free media, and BSA 10ml/mg. We then incubated the plate for 30 min at 37C and 5% CO2. After the incubation we removed the media from the cells and added 150Â µl of SDS ample buffer with othovanadate, pipetting up and down to remove the cells. We then transferred the cells to microfuge tubes and placed them in a water bath, boiling, for 5 min. We then preformed an SDS PAGE with six wells, the first five we ...