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Dopamine Active Transporter

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Dopamine & DAT (Dopamine Active Transporter)
The central dogma of biology involves the transcription of DNA to mRNA and the subsequent translation of the mRNA to protein. This experiment focused on identifying the Dopamine Active Transporter, commonly abbreviated as DAT. DAT is a consists of 12 transmembrane domains. It is responsible for mediating the reuptake of dopamine from the synaptic cleft back into the cytosol. With dopamine being one of the key neurotransmitters, it is associated with a few coming illnesses, one of which is that of Parkinson’s disease. Parkinson’s disease is the gradual degeneration of the dopaminergic neurons. As a treatment method, a precursor of dopamine is introduced to patients in the form of L-DOPA. In order to synthesize the dopamine, the amino acid tyrosine is converted to L-DOPA via the enzyme tyrosine hydroxylase and the L-DOPA subsequently converted to dopamine via aromatic amine decarboxylase. The movement of DA and its synthesis is summarized in Figure 1. After the successful transcription and translation of DAT, it is trafficked to the surface of the cell. A common way to determine whether or not the transporter was successfully transported to the surface, is immunolabeling, which …show more content…
In Human Embryonic Kidney (HEK) cells, encoded DAT genes attached to Yellow Fluorescent Protein (YFP) genes, with the use of a plasmid, created DAT and YFP proteins. Fluorescence in the cell was caused by two antibodies. Materials.
Conducting this experiment required the use of Phosphate-Buffered Saline (PBS), 3.7% paraformaldehyde in PBS (Fixing Solution), PBS + 10% FBS + 0.1% triton X- 100 (blocking agents), 250ul/coverslip Rat Anti-DAT, 1:750 , 250 ul/coverslip Alexa 546 Chicken Anti-Rat-DAT 1:1000 , DMEM (supplemented with 10% FBS and pen-strep, 0.25% trypsin solution), T-25 flask, 6-well plate, and a CO2 incubator.

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