5 Flashcards
(108 cards)
most important factor for deciding how to purify protein
cellular localization
why do you need to purify proteins
other molecules/ions/proteins may fuck with or modify ur protein, creating a heterogenous population
first step to understanding protein function
purification
what question do you ask yourself in order to know where to get your crude extract for purifying proteins
where am i getting the protein from? choose correct cell or tissue source is needed to get an adequate quantity and quality.
how can molecular biology help you get crude extract
genetically engineer protein of interest
intracellular protein crude extract generation extra step
lysing cell
physical methods of lysing cell
grinding, sonication, vortexing with glass beads
other non physical methods of lysing cell
osmotic pressure, chemical basis (detergents)
why is it bad to use osmotic pressure for lysing cells
interact with water, forming H bonds, denaturing protein (that’s why you need buffer, to preserve structure, when doing lysing)
how to get rid of the other shit in crude extract
centrigufation to separate supernatant from pellet of large organelles or insoluable. the bottom of the container will be pellet. this can also be used to isolate a particular organellle or cellular compartment if you know where it is
ultracentrifugation used to isolate what
used to isolate larger protein complexes
chromatography
differential particioning of molecule between mobile (buffer) and stationary (column) phase
protein absorbance
biomolecules absorb light at a specific wavelength
what measures protein absorbance
spectrophotometer
do most proteins absorb visible light
no
how does absorbance indicate concentration of protein in solution
absorbance at 280 nm from aromatic amino acids. the aromatic ones can absorb and relase light
how can stains be used to visualize or quantify proteins
coomassie blue can bind noncovalently, irreversibly for a bradford assay
beer lambert law common formula
molar extinction coefficient (depending on trp and tyr number) times concentration times path length of cuvette
beer lambert law log formula
log(light that went through/light transmitted)
size exclusion chromatography/gel filtration
column with resin of porous beads that allow small proteins to enter while large ones don’t go in. elongated may appear larger due to orientation
why do you need calibration with known MW proteins for size exclusion chromatography
because then you know the size of the unknown
void volume
size exclusion. anything larger than the fractional range of the column goes straight through.
elution volume
size exclusion. when the protein first starts to come out
total volume of column
size exclusion. Vt