Prion Protein Lab Report

Introduction Cellular prion proteins are normally occurring glycoproteins found on the outer surface of neuronal cells. They are also expressed by most other cells found in the body. Prion proteins are attached to the plasma membrane by a C-terminus glycosyl-phosphatidylinositol (GPI) anchor. The prion protein exists in two conformational states: a cellular α-helix-rich isoform (PrPc) and the prion disease-associated β-sheet isoform (PrPsc). In humans, PrPc is composed of 253 amino acids and is encoded by the PRNP gene, which is located on chromosome 20 (Adams et al, 2014). The N-terminal domain of the protein contains a highly conserved octapeptide repeat (OR) sequence made up of a PQGGGWGQ peptide sequence. The octarepeat contains histidine

In this experiment, the specificity of the assay for prion proteins will be determined. It is hypothesized that a chemiluminescent probe will react with reactive oxygen species (ROS) to produce an excited state (CLP-O) and hv. The light (hv) produced is detected with a photomultiplier. The assay will be evaluated using cell lines cultured in the presence and absence of siRNA. The siRNA acts as a construct that causes the inhibition of the PrPc expression in the PRNP gene. Further characterization of lymphocytes for protein expression will be conducted through monoclonal antibody specification by means of Western blots and Flow Cytometry. If the assay proves to be specific, the process of characterizing cells will become more efficient. If the research established a conclusive connection between healthy controls, Chronic Fatigue Syndrome patients, and their dysfunctions in controlling oxidative stress future development of diagnosis and treatment tools will be

The cells/stain solution was loaded into a Countess™ cell counting chamber slide which was inserted into the Countess™ Automated Cell Counter. If the live cell count was above 1 x 106 cells/mL, then the 1mL of cells, previously taken from the culture flask, was washed 1x with 14mL PBS and centrifuged for 8 minutes at 1200 rpm. The supernatant was aspirated off. The pellet was resuspended in 1mL PBS and then placed on ice. The following reagents were thawed while the cell count was performed: yellow fluorescent chemiluminescent probe (CLP), activator solution and