Prion Disease

Why is the secondary antibody used in an ELISA test conjugated with an enzyme? What happens when this enzyme meets up with its substrate?

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

Bradford assay would be ideal if an accurate estimation of a newly isolated protein using BSA as a standard. With the data obtain from the Bradford assay, it shows that it was more sensitive and quicker, also it had less interference from substance that were possibly present in the protein solution, expect for the interfering substance SDS.

Proteinaceous Infectious Particles, commonly known as Prions, are extremely rare misfolds of the protein PrPc, which cause fatally neurodegenerative diseases, and are theorized to be infectious only by the protein itself (U.S National Library of Medicine, 1998). This “protein-only theory” is still heavily debated today, as some scientists deny the theory, and there isn’t a significant amount of evidence on each side to qualify the theory or disprove it (Soto, C. 2011). The base “Prion” protein is encoded in the gene PRNP, while being non-infectious. Prions are most commonly found in human prion diseases, but they can also be in other animals in the form of Mad Cow Disease and Chronic Wasting Disease, classified as Bovine Spongiform Encephalopathies

This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.

Gel electrophoresis method. Qualitative analysis shows protein concentrations in kidney, heart, and liver. 1-6 are kidney tissue. 7-14 are liver and 15-19 are the heart tissue.

The purpose of this lab is to test for enzyme activity by examining factors that may influence enzymes.

ELISA works on the principle of an antigen binding to specific antibody (lock and key), which can be used as a way to identify quantities of proteins in a small sample of fluid. The specific proteins used in an ELISA are estimated quantitatively. The ELISA test is carried out by incubating the serum that contains the antigen of interest with antibody’s within a well, in order for the antibody’s to bind with the specific antigens. The plate is then washed with a mild detergent in order to remove any proteins that have not been bound. The washing of the plates is carried out between every step in order

The tests that need to be done following an oxidase positive test are lactose fermentation, hydrogen sulfide production, and microgen A and B test. These test need to be done together in order to speed up the process. The lactose fermentation test is done in a phenol red broth and is used to determine if the organism ferments lactose. Results of this test can be seen in figure 5.

There are two methods using which the BCA Protein Assay can be performed- Test tube method and Micro plate method.

In addition to the cervid species in which chronic waste disease can occur in wide variety of other animals. These other animals include monkeys, sheep, cattle, prairie voles, mice, and ferrets. Environmental transmission has been linked to contact with infected bodily fluids and tissues. Once the prions are in the environment chronic waste disease prions may remain infectious for many years. Thus, Decomposition of the disease carcasses, infected gut piles left by hunter, as well as urine, saliva, feces, and antler velvet of infected individuals that are deposited in the environment.

Prions are a type of protein found naturally in the brain and other regions of the central nervous system. The diseases associated with

Prions are an incongruous pathogen that is causative of transmissible spongiform encephalopathies (TSE); a group of fatal neurodegenerative diseases found in mammals. This abnormal proteinaceous particle is believed to be a post-translational mutation of a normal cellular protein found in the brain (Emmanuel, 2002). This naturally occurring pathogen acts similarly to a viral infection, however as it lacks nucleic acid it is invulnerable to death via means of antibiotics or sterilisation. PrPc and PrPsc, the known prion proteins indicative of TSE’s, are known to bind to the surface of neurons and fold these proteins into an abstract spongiform structure. Although the process inducing this folding mechanism is unknown, it is

The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.

each O-Ring of this array has capacity to capture much more than the critical mass of target analytes above the detection limits of ELISA and this dramatically improves the reliability and accuracy of each ELISA,