BMS tools Flashcards
Her2
Human epithermal growth factor 2
- for 1 of every 5 breast cancer, cancer cells make an excess of HER2
Epitope Tags
added to a protein through recombination techniques
epitope is the area of the molecule where the antibody binds
this is useful if a good antibody can’t be produced
Two uses of immunoaffinity chromatography
-you run cell extract over beads, affinity purification: you want to isolate the protein of interest from extracts
isolation and identification of binding proteins - you have your protein of interest attached to a bead, you run lysate over it. You then elute and dissociate, run a SDS page gel. Cut out gel and sequence the protein.
What is immunohistochemistry?
You use antibodies to target and label specific proteins IN TISSUE.
You then add a secondary enzyme that binds to an conserved region of every primary antibody that will undergo an enzymatic reaction when given substrate. You can then localize the antibody/enzyme complex.
MDM2
- an oncoprotein (a protein made by an oncogene which promotes tumor formation in high levels)
- in response to cellular stress MDM2 promotes p53 ubiquination and degradation.
Co-immunoprecipitation
Looks like its like immunoaffinity chromotgraphy, focused only on binding partners where as the other could be a purification technique.
So you take antibody to your protein of interest, you isolate the protein from you sample. You purify and use SDS page to separate the partners. Do a western blot using an antibody to suspected binding partner or cut and sequence. If its there it establishes interaction. clinical example: p53 and MDM2
Troponin
Highly specific for myocardial injury, not elevated in trauma,
A patient has a positive HIV ELISA test, what does that mean? What is next step?
Patient most likely has HIV, order a Western blot
What is the criteria for HIV western blot tests?
One of bands p24 or p31
One of bands gp120 or gp160
ELISA
Enzyme-Linked Immunosorbent Assay
Purpose: detect antibody or antigen (troponin)
Traits: highly sensitive, need little sample but because of it, less specific. The golden standard in HIV testing is Elisa first (if negative its over) followed by Western blotting.
Example: we know the proteins to HIV, you link antigens to the plate, then you add sample and wash nonbinding partners off. Add a secondary antibody and color develops, then you analyze.
Home pregnancy test.
Window Period (HIV)
4-6 week period between infection and when the antibodies to HIV can be detected.
Indicator
Antibody with bound enzyme that convert colorless molecules to colored ones.
Example: alkaline phosphatase
Horseradish peroxidase
Western Blot
Specific to proteins,
you run an SDS page, then you run a current through the gel with one side in contact to nitrocellulose membrane which is positive and binds everything.
Then you can expose your isolated proteins to a primary antibody and subsequently a secondary one.
How does one develop primary antibodies?
Lets say you want to isolate a bunch of rabbit antibodies. You take a solution with the antigen of interest. You inject a rabbit with it. And the rabbit is immunized. You drain the rabbit QQ, take the serum, isolate the primary antibodies from it.
Erythropoietin
used to treat anemia, it increases Hb, maximal oxygen uptake and the time it takes till exhaustion.
The use of EPO by atheletes (blood doping is inhibited. You can run an SDS page and against a ladder of normal and the erythropoetin from drugs.
