Lecture 20 - Biochemical Techniques Flashcards
what kind of physical/chemical characteristics can be exploited to learn more about molecules(proteins)?
- size
- charge
- specificity
- solubility
why do we want to purify proteins?
- to understand the structure
- to understand the mechanisms of action
- to detect protein modifications
- to identify protein binding partners
def: rupturing the cell to release the internal contents
homogenization
what kind of cells need to be broken to get enough protein
- tissue from an organ
- tissue culture cells
- yeast cells
- bacteria cells
what do we want our cell homogenate to have?
- disrupted the plasma membrane
- released soluble proteins
- maintain the integrity of organelles
Lysis buffer needs to help break apart the cell, but not all the ___________
organelles
what is frequently added to lysis buffers to prevent protein degradation
protease inhibitor
what is the disadvantage of using harsh lysis buffers, like amphipathic detergents?
they can denature the protein
what are the two common techniques to fractionate a sample?
- differential centrigfugation
- density gradient centrifugation
different __________ __________ separate organelles or macromolecules
sedimentation rates
def: separation of cell organelles or other particles of different size and density by their different rates of sedimentation in a centrifugal field
differential centrifugation
T or F: differential centrifugation does not yield a pure sample, so further steps are required
true
def: a measure of how rapidly the particle sediments when subjected to centrifugation, expressed in terms of Svedberg units
sedimentation coefficient
def: more contaminants can be removed using this kind of centrifugation, samples are placed on top of a gradient, centrifugation causes particles to move into discrete zones, based on the density of the gradient and the density of the particle/organelle
density gradient centrifugation
dense particles move into the gradient ______ __________ than less dense
more rapidly
if you want a specific protein, what do you need to know?
you need to know its properties to be able to identify a specific protein
def: a procedure where a mixture of dissolved components is fractioned as it moves through some type of porous matrix (usually in a column)
chromatogaphy
differential and density gradient centrifugation give us relatively pure peroxisomes, but if we want to further purify them, what do we do?
use in enzyme assays
what are the 3 types of chromatography?
- ion-exchange chromatography
- gel filtration chromatography
- affinity chromatography
def: relies on the differences in surface charge of the macromolecule, beads carry a weak (+) or (-) charge, charge proteins interact with beads to varying degrees, and proteins can be eluted using solutions at characteristic pH and salt concentrations
ion-exchange chromatography
proteins can be eluted, come out the bottom of the column, by doing what?
washing the column with different pH buffer
what happens as the pH is lowered in ion-exchange chromatography?
- negatively charge amino acids become neutral
- positively charged amino acids become more abundant
def: relies on difference in the size and shape of the macromolecules, beads have different size pores that allow proteins to percolate through them, smaller proteins enter all the pores and take longer to get through the column, larger proteins pass through the column more rapidly
gel filtration chromatography
def: relies on highly specific interactions between a ligand/substrate/antibody and the target, beads have a specific molecule attached to their surface and preferentially bind to the protein being purified, proteins are unbound and purified using salt, a change in pH , or mild detergents
affinity chromatography
