Lecture 8 - Purification, Detection, and Characterization of Proteins II Flashcards
Provide a summary of mass spectrometry.
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of proteins. You can subject a protein to trypsinization (cuts at specific amino acid sequences) and run it through a chromatographic column. The eluted peptides are formed into a high pressure aerosol and spit into the MS machine. They become ionized and in the analyzer, they are ejected and will fall down on a detector as a function of their weight (heavier first, lighter later) such that a mass to charge ratio can be calculated. The MS machine will make a fingerprint of all the peptides that were formed and tells you which protein you had.
What is isolated in glutathione S-transferase (GST) affinity chromatography?
Glutathione
What will nickel chromatography columns separate proteins by?
Nickel chromatography columns will separate proteins by histidine content, since it is an amino acid that will interact with metals.
How are immuno-affinity chromatography columns prepared?
Protein A is a protein that interacts with the FC domain of an antibody. You can conjugate purified protein A to agarose, and use that to interact with antibodies. The antigen recognizing portions of the antibodies then bind the antigen when the column is run.
Provide a summary of immunoprecipitation.
IP allows you to isolate a small amount of protein from a complex mixture. You add the proteins antibody to your mixture, followed by protein A conjugated beads. You then perform a simple centrifugation to precipitate the beads bound to the antigen protein (through protein A and the antibody) at the bottom of the flask.
Provide a summary of co-immunoprecipitation.
Co-immunoprecipitation is an immunoprecipitation that is a bit more gentle and that brings down all the proteins that may be interacting with your protein of interest.
How do you antibody stain a sample?
You would take a sample of proteins, incubate them with an antibody, and then apple a second antibody that is conjugate to a fluorochrome (a specific dye that, when given a wavelength, emits a longer wavelength while starting to deexcite and become fluorescent).
What is a fluorochrome that emits red light?
Rhodamine
What is Martin Chalfie known for?
GFP
Where does GFP come from?
It comes from the jellyfish Aequorea victoria.
Why is GFP so beneficial?
GFP is great because it can be attached to any protein and be able to follow that particular protein in the cell so that we can see what’s happening.
