Download Western Blot & ELISA Techniques: Antibodies, Blocking, Primary & Secondary Antibodies and more Quizzes Biology in PDF only on Docsity!
western blot (immunoblot)
technique that allows one to detect any quantify proteins that react with a specific antibody. Used to determine if a given antigen is present, and concentration of the antigenic protein. Useful for medical testing (HIV, other viruses, Mad cow's disease, lyme disease) but much as been replaced by PCR TERM 2
antibody
DEFINITION 2 protein that binds to a specific sequence of amino acids, or epitopes. They have specificity which is the ability to bind to specific target with low background, and affinity, which is how tightly it binds to the target TERM 3
polyclonal antibodies
DEFINITION 3 produced by injecting proteins into animals, which are specific to multiple epitopes on a protein. THey have lower specificity, higher affinity, and are more tolerant of different fixation conditions TERM 4
monoclonal antibodies
DEFINITION 4 produced from fusing B-cells with cancer cells, target a single epitope. They have high specificity, lower affinity, and may be sensitive to fixation conditions for tissue specimens. TERM 5
nitrocellulose or PVDF (polyvinylidene
difluoride)
DEFINITION 5 On the SDS page after the gel is removed it is placed on this flat surface. It is less expensive and does not need to be activated by methanol, but PVDV has higher binding capacity and is reusable if desired.
blocking
the membrane has free sites that is coated with a mixure of nonspecific proteins to block these free sites in this process. Powdered milk is a good one to use TERM 7
primary antibody
DEFINITION 7 after blocking then the membrane is soaked in a solution with this to the protein of interest which can be monoclonal or polyclonal. The antibody can adhere to the membrane only if it interacts with its specific antigen TERM 8
secondary antibody
DEFINITION 8 most frequently used way to detect a primary antibody bound to a specific protein is to introduce this that will react with any antibody from the same biological source as the primary antibody. You inject the constant Fc region of an antibody into another species. This antibody can be covalently coupled to an enzyme that catalyzes a chromogenic reaction or alkaline phosphatase. It can also be assayed by immersing the membrane into the substratie of the coupled enzyme. This enzyme is very useful as it will target any antibody from the primary source TERM 9
enzyme-linked immunosorbent assay (ELISA)
DEFINITION 9 similar to western blotting and allows for the detection of an antigen or antibody in a sample. Used as a quick medical diagnostic tool to detect the presence of certain protein markers or viruses in a patient, industry to check sample quality, and in pregnancy tests. Identical to western blot except for SDS-PAGE gel is not run, instead samples are placed in small wells and assayed. If reaction occurs then well changes color which means that antibody has bound to an antigen. Disadvantage is it provides no information on the molecular weight of an antigen. An antibody on ELISA could bind nonspecifically and it would be difficult to detect TERM 10
indirect ELISA
DEFINITION 10 has a setup identical to the western blot in that the antigen is on the membrane which is bound by a primary antibody followed by a secondary antibody coupled to an enzyme
