1. eukarotic cells tools and techniques Flashcards
cell theory
cells are fundamental units of life
all organisms are composed of cells
all cells come from pre-existing cells
all cells are surrounded by a plasma membrane
typical size of ourcells
20um
size of prokaryotic cell
1um
thermos aquiaticus is an example of
an archaea prokaryote
all eukaryotes have 3
a nucleus, cytoskeleton and complex internal membrane systems
model cell system for prokaryote, lower eukaryote and high eukaryote
e. coli
yeast s.cerevisiae
human tissue culture cells HeLa
model animals 3
zebrafish for vertebrate development
drosophila for genetics
nematode worm for genome sequencing
function of peroxisomes
break down fatty acids, alcohols and toxins
Zellweger syndrome
caused by defects in making new peroxisomes
is an imbalance in lipid mtablosim
nucleolus
rRNA transcribed here and ribosomal units assembled here
endoplasmic reticulum
entry point to secretory pathway
makes secretory proteins, membrane proteins and lipids
smooth ER
is abundant in human cells involved in lipid metabolism and in the liver for detoxification of lipid soluble compounds
sarcoplasmic reticulum
is a ER derived calcium store in muscle cells, it has an important role during muscle contracting
golgi
receives proteins and lipids as cargo from the ER
modifies cargo eg glycosylates proteins and sorts cargo to correct tlocation
endomembrane system
nuclear envelope, endoplasmic reticulum and Golgi evolved from the plasma membrane
where oes protein synthesis occur
in the cytosol
microscopy
study of tissues cells subcellular compartments in a buiological context
centrifugation is the isolation of subcellular organelles
chromatography is the purification of proteins and protein complexes
gel electrophoresis, mass spectrometry
is the analysis of macromolecules proteins and DNA
light microzopy
descriptive differences
live, chemically fixed cells used (preserved and stabilised for microscopy)
DAPI and Hoechst
pluroescent dyes/probes which preferentially bind to DNA
transmission electron microscopy
thin specimen section stained with heavy metals for contrast
detailed subcellular structure
scanning EM
good for 3D images
homogeniation
controlled rupture of plasma membrane, releases intact organelles
centrifugation
homogenate is centrifuged, results in supernatant fluid containinf smaller and less dense components
pellet containing larger more dense components
differential centrifucation
repeated centrifugation at progressively high speeds , fractionates the contents
low speed= whole cells nuclei cytoskeletons
med speed - mitochondria lysosomes and peroxisomes
high speed- closed fragment of er, small vesicles
v high speed - ribosomes and viruses
velocity sedimentation
use sucrose gradient to get highly purified organelles eg ER and mitochondria
SDS page
proteins have negatively charged detergent SDS bind to it to unfold/ denture it
reducing agent agen t to proteins to cleave the disuplide bonds
proteins separated by size. smaller= faster
SDS page stainss
conassie blue
silver staoon
radiolabel
immunoblotting
identify a specific protein using an antibody that recognises it
Blot the SDS PAGE by transferring onto nylon sheet
peptide mapping
used to identify unknown proteins ina gelslice without using antibodies. use mass spectrometry
gel filtration column chromatography
separates proteins by size. larger proteins pass through column more quicly
ion exchange chromatograpphy
separates prtoeins by charge
