KH7 Flashcards
Describe chromatography
Separation of a sample based on how its components interact with an immobile/solid phase (chemical properties).
What’s gel filtration chromatography?
The solid phase is composed of gel beads with molecular pores that allow smaller particles to flow right through them (faster).
What’s ion-exchange chromatography?
The solid phase is composed of charged gel beads that will attract oppositely charged particles. The bounded molecules are then released by flowing a salt solution (get exchanged with Na+ or Cl-).
Describe what antibodies are and how they function?
They’re proteins that recognize specific antigens by binding to an epitope (molecular target).
True or False? “Only a few types of antibodies exist”
False. Each antibody has one epitope but the amount of unique targets leads to a practically infinite number of antibodies.
How can an antibody only bind to a specific protein in presence of many more?
If only that protein possesses its epitope.
What’s antibody-affinity chromatography?
The solid phase is composed of antibodies looking for their epitope. The target protein can be released by lowering the pH (washing).
True or False? “Other than the CDR, every species shares the exact same chemical structure for their antibodies”
False. The heavy and light chains chemically vary from one species to the next. However, only the CDR changes within a species.
What’s an immunoblot (western blot)?
Recognition of proteins (through antibodies) initially separated by SDS-PAGE.
Describe the steps of an immunoblot
After the electrophoresis, an electric field pushes proteins onto new membrane (prints) which gets into contact with Ab1, then Ab2. The Ab2 comes with a tag: either fluoresces or generates a specific product.
What’s immunoprecipitation?
Separation of a specific protein from a mixture through antibody binding. An immunoprecipitate (targeted protein, Ab, “scaffold” protein) is collected.
What’s co-immunoprecipitation?
Target proteins will sometimes exist in a complex with other proteins. These partners also get collected once the target protein binds to the Ab.
What’s immunofluorescence microscopy?
Same process as immunoblot. Directly shows protein distribution in an organic environment.
True or False? “Multiple proteins can be studied simultaneously using immunofluorescence microscopy”
True. Just need to raise new primary and secondary antibodies for each (could come from same or different species).
What’s the advantage of using GFP?
It allows to study living cells (everything else requires to kill the subject): used as a reporter gene or fusion protein.
