Chapter 8: Analyzing Cells Flashcards
Method to study protein encoded in RNA
use bacteria (they’re simple)
Method to study function of organelles/intracellular functions
use eukaryotic cells
don’t need something more complex, such as context in a tissue
cells can be isolated from tissues
most realistic
1) disrupt extracellular matrix and cell-cell junctions (use proteolytic enzymes to digest proteins and chelator agents)
2) tease apart tissue into cells by gentle agitation
methods to break open cells (don’t destroy the inside!)
high frequency sound, detergent (pokes holes in membrane), force cells through small tight spaces so they burst, glass vessel and pestle to shear open
what do you end up with after breaking open cells?
a homogenate
homogenate
mix of membrane components, cytoplasm, proteins
cells can be grown in culture
aka in vitro (in glass)
can culture plant and animal cells
purifying protein step one
extraction from inside the cell (break cells open)
centrifugation
rotates cell extracts at high speeds
separates them by size and density
impure, multiple centrifugations can improve purity
centrifugation where do larger objects go
large object = most centrifugal force = pellet at bottom
low speed centrifugation
pellet contains whole cells, nuclei, cytoskeletons
medium speed centrifugations
pellet contains mitochondria, lysosomes, peroxisomes
high speed centrifugation
pellet contains microsomes, small vesicles
very-high speed centrifugation
pellet contains ribosomes, viruses, large macromolecules
supenatant
liquid above pellet
contains lighter molecules
microsomes
resealed vesicles formed from fragments of rough and smooth ER
column chromatography
separates by interaction w/ matrix
ion exchange chromatography
separates on charge
resin is positive, positive flows through
can elute with diff concentrations of salt
hydrophobic chromatography
hydrophobicity
column contains beads with hydrophobic chains, retard hydrophobic
gel-filtration chromatography
size
large go through first, small get stuck in pores of beads
helps estimate size
affinity chromatography
ability to bind to particular small molecules or to other macromolecules
EX: bead covered in polysaccharide, enzyme that operates on it will be retained
later can be washed off in pure form
EX: antibodies
high-performance liquid chromatography (HPLC)
silica based resin
separates even at fast flow rates
great choice!
protein purification by chromatography requires:
multiple steps in succession (usually)
a functional assay to follow purification so you know what fractions to keep
