Isolating & analyzing proteins Flashcards
what is subcellular fractionation?
process of isolating cellular components whilst keeping their functions intact
what is the most distinctive property of a protein that can be used to separate it?
function
What are the two most common ways to separate proteins based on size and charge?
size exclusion chroma and ion exchange chroma
what is size exclusion chroma?
column packed with porous polymer beads, protein run through using solvent and separated out based on size. Large come out first, smaller partition in out more
what is ion exchange chroma?
uses a charge at a particular pH, beads are either positively or negatively charged
things that don’t stick to column elute first
add salt to make positive charged proteins be dislodged
What is affinity chroma?
if the function of the protein is known and what helps it perform that function an affinity tag can be made which is then put on beads. The unwanted proteins are then washed off because the substrate etc… is bound to the beads. The protein of interest is washed off by using a ligand solution
best method
Can you add an affinity tag to a recombinant protein for expression and purification?
YES
what happens in electrophoresis? (vague)
charged proteins are separated based on their different mobilities in an electric field
what does SDS-PAGE stand for?
SDS polyacrylamide gel electrophoresis
what does SDS-PAGE separate based on?
negative charged sds-protein micelles based on their size and thus their molecular weight
in SDS-PAGE what size proteins migrate fastest toward the + electrode?
small!!
why is a reducing agent added? (sds page)
to cleave S-S bonds and resolve individual polypeptide chains
What is a 2-D page?
separates by both pH and size (1 d ph, 2 d MW)
can reveal hundreds of proteins and disease related difference in protein levels
Can proteins be sequenced using mass spectrometry?
YES. how fast they fly depends on their charge and inversely on their mass
what is trypsin used for in mass spectrometry?
to cleave the peptide bonds at lys and arg
what is peptide mass fingerprinting?
use trypsin to cleave and then get a mass spec which you use to compare to the theoretical masses for all trypsin released proteins in a data base
what is tandem MS-MS?
after trypsin digestion mass spectrometer gives peptide masses which are then further fragmented at their peptide bonds. Once fragmented more a coupled spec is created giving the fragmented masses. The mass difference between fragments can be used to determine a partial AA sequence which can then be used gene identification or gene cloning
What tool determines the 3D arrangement of atoms in a protein starting of with a crystal?
X-ray crystallography
hit crystal with at varying angles with x rays to get a diffraction pattern. You can then work out the electron density map which provides details on conformation binding partners and enzyme function
What tool has contributed the most structures?
Xray crys.
What is a problem with x ray crystallography?
get a somewhat static view of the proteins structure because it is in its crystal form which is not representative of how it behaves in aqueous solution
what does NMR stand for?
nuclear magnetic resonance
what is a benefit of NMR?
don’t need crystal form of protein–works in solution
but it has to be pure and all that is present in that solution
what nuclei spins are measured in NMR?
1H, 13C, 15N
what can the electromagnetic resonance of frequency of nuclei used for?
to provide information on the neighbouring atoms, through bonds or space
What does NMR generate?
the possible conformations of the protein that fit the data–not just one answer
tells you what parts of the protein are flexible which has to do with function
only found 10% of structures this way
Why do we want to solve the 3-D structure of proteins?
tells us about function
tells us how mutations may affect function
development of drugs–create molecules that inhibit a particular enzyme in vitro
