Dopamine Lab Report

Locating the Dopamine Transporter via Immunostaining and Colocalization

Taryn Nurse+, Kadija King+, Dasia McClain+, Jahmel Fowler+, Phyllis Freeman+, and Shawn Goodwin*.

Life and Physical Science Department, Fisk University+ and Department of Biology. Meharry Medical College School of Graduate Studies and Research*, Nashville, TN, 37208.

Introduction (Dasia)

The focus of this experiment was to identify the Dopamine Active Transporter (DAT), known as SLC6A3, which is located on the 5th chromosome. By using a plasmid, the gene that encodes for DAT was attached to the gene for a Yellow Fluorescent Protein (YFP) in Human Embryonic Kidney (HEK) cells. This resulted in the production of the DAT and YFP proteins in the cells. Two antibodies were used to cause fluorescence in the cells.

Background (Taryn)

Dopamine Active Transporter (DAT)

The DAT is a 12-membrane spanning protein that clears away excess dopamine from the synaptic cleft between neurons.

Dopamine

Dopamine is a vital neurotransmitter found in dopamine neuron.

Dopamine acts to inhibit acetylcholine, which is responsible for muscular contractions. The gradual loss of dopamine is attributed to the abnormal nerve-firing patterns associated with Parkinson’s disease. Parkinson’s disease is a progressive disease caused by the deterioration of the central nervous system. In treatment, dopamine is presented to patients in the form of a precursor, L-DOPA. Dopamine is biosynthesized from one of 20 the essential amino acids. This occurs when tyrosine is converted to L-DOPA with hydroxylase. L-DOPA is then converted to dopamine via a decarboxylation

reaction.

HEK cells (Jahmel)
HEK 293 or Human Embryonic Kidney cells 293, are a cell line derived from human embryonic kidney cells usually grown in tissue culture and used for lab experiments.

Colocalization (Jahmel)
In order to perform the experiment, antibodies were used to tag the DAT. Two different antibodies were used, as each would fluoresce a different colour. Areas in which co-localization or where both fluorescent colours were noted would indicate the location of the DAT. A primary antibody was assigned to the YFP-DAT by injecting the purified protein into a rat, which resulted in the production of an antibody that recognizes the DAT, known as rat-anti DAT. The Alexa antibody was then purified and injected into a chicken, resulting in a secondary antibody that recognizes the antibody created by the rat, known as chicken-anti rat-anti DAT. This protein would fluoresce red on its own when excited by light with a wavelength of 562 nm and the first would fluoresce green after exposure to light with a wavelength of 488 nm. Colocalization would be identified by yellow fluorescence.

https://www.researchgate.net/profile/Dale_Edmondson2/publication/7227442/figure/fig2/AS:267630162477070@1440819411229/Figure-1-Dopamine-synthesis-and-its-metabolism-by-MAOA-and-MAOBThe-pathway-of-dopamine.png

Materials and Procedure (Kadija). Immunofluorescence Staining of Formaldehyde-fixed Adherent Cells. This experiment required the use of PBS (Phosphate-Buffered Saline), 3.7% paraformaldehyde in PBS (Fixing Solution), PBS + 10% FBS + 0.1% Trition X 100 (Block/Permeabilization Solution), 50 µl for parafilm Rat Anti-DAT 1:750 (Primary Antibody), 50 µl for parafilm 1:1000 Alexa 488 Chicken Anti-Rat (Secondary Antibody), and a 12-well plate.

Fixation. Due to convenience, cells grown on a small round coverslip were fixed and stained in a 12-well plate. A total of 250 µl/cs (50 µl/cs for parafilm) was used for the antibodies. The cells were fixed in 3.7% PFA diluted in PBS for 15 minutes at room temperature, and a volume of 0.5 ml/well of a 12-well plate. The PFA was aspirated and rinsed gently 2 times with PBS. Due to holding for immunostaining, the PBS remained in the well with 0.001% sodium azide.

Immunostaining. The cells were incubated with blocking permeabilization solution for 30 minutes at room temperature. The blocking permeabilization solution aided in dissolving the lipid’s bilayer which increased penetrability to antibodies. Next, the cells were incubated with primary antibody solution for 30 minutes at room temperature. A total of 50 µl/coverslip was used on the parafilm. The coverslip was placed back into the 6-well plate. After incubation of the primary antibody sample, excess was washed 3 times per 5 minutes with PBS. Then the secondary antibody solutions were incubated for 30 minutes at room temperature. A total of 50 µl/coverslip was used on the parafilm. The coverslip was placed back into 6-well plate. Excess of the secondary solutions were washed 3 times for 5 minutes with PBS. Lastly, the coverslip was inverted onto a drop of mounting media on a labeled slip and sealed with self-sealing resin; the self-sealing resin was allowed to dry before fluorescence microscopy.

Data

Acknowledgements. (Kadija) Shawn Goodwin, PhD, Meharry Medical College, Phyllis Freeman, PhD, Fisk University