AcLlvlLy 7

lsolaLlon of MlLochondrla and

nucleus
krlsLlne A 8evllla
aulo Sallbay
Ma LsLher SamarlLa
nlcole Marle u Sembrano
Claudla SeLlawan
Cell lracLlonaLlon
W A process where ln Lhe organelles can be
removed from cells and be separaLed from
one anoLher whlle sLlll malnLalnlng Lhe
properLles of Lhe lndlvldual cell
W 1he fracLlonaLlon of cells lnLo organelles
always beglns wlLh Lhe dlsrupLlon of Lhe cells
Lo release Lhelr conLenLs lnLo a benlgn buffer
W 1he resulLlng maLerlal ls called Lhe ce||
homogenate or Lhe ce|| extract
rlnclples of cenLrlfugaLlon ln Lhe
lsolaLlon of sub cellular parLs
W 1he mosL common way Lo perform cell
fracLlonaLlon ls Lo dlsrupL Lhe cells and separaLe
Lhe subcellular componenLs by centr|fugat|on
W arLlcles sub[ecLed Lo Lhe acceleraLlon of a
cenLrlfugal fleld wlll move or sedlmenL aL a
veloclLy deLermlned by varlous properLles of Lhe
parLlcle lncludlng lLs mass lLs relaLlve denslLy
(compared Lo LhaL of Lhe suspendlng fluld) and
lLs frlcLlonal properLles (shape)
uenslLy CenLrlfugaLlon
W SeparaLlon ls achleved
based on Lhe slze of Lhe
parLlcles ln dlfferenLlal
cenLrlfugaLlon
W 1hls Lype of separaLlon ls
commonly used ln slmple
pelleLlng and ln obLalnlng
parLlallypure preparaLlon
of sub cellular organelles
and macromolecules
MlLochondrla
W A membraneenclosed
organelle found ln
mosL eukaryoLlc cells
W lL ls responslble for Lhe
chemlcal reacLlons ln
Lhe producLlon of
energy ln Lhe form of
A1 as a resulL of
oxldaLlve
phosphorylaLlon
nucleus
W A mebrane bound
enclosed organelle found
ln eukaryoLlc cell
W lL conLalns mosL of Lhe
cell's geneLlc maLerlal
organlzed as mulLlple long
llnear unA molecules ln
complex wlLh a large
varleLy of proLelns such
as hlsLones Lo form
chromosomes
W lL ls known as Lhe conLrol
cenLer of Lhe cell
A reparaLlon of speclmen for
cenLrlfugaLlon
A LoLal of 20 grams of Lhe ouLer 2 3
mm of Lhe ouLer caullflower surface was
cuL
2 Caullflower Llssue was placed ln a
chllled blender wlLh 40 ml of lce cold
manlLol grlndlng medlum 1hen
homogenlzed for 3 mlns
3 1he sample was fllLered wlLh Lhe use
of a cheese cloLh 1he fllLraLe was
Lransferred lnLo a chlled 30 ml
cenLrlfuge Lube
8CCLuu8L 8 CenLrlfugaLlon
*8e acqua|nted w|th the funct|on and operat|on of the refr|gerated centr|fuge
w|th the he|p of your |aboratory |nstructor
Carefully decanL Lhe supernaLanL (uone ln arL A)
lnLo a fresh chllled 30mL cenLrlfuge Lube Spln aL
2000 rpm for 0 mlnuLes aL 4C
uecanL Lhe supernaLanL lnLo a clean chllled
cenLrlfuge Lube and cenLrlfuge aL 0000 rpm for 20
mlnuLes aL 4C (nuCLLA8 LLLL1)
uecanL Lhe supernaLanL lnLo a clean chllled cenLrlfuge Lube and
cenLrlfuge aL 3000 rpm for 20 mlnuLes aL 4C Add 30mL of
Lhe lcecold mannlLol medlum Lo Lhe pelleL (Ml1CCPCnu8lAL
LLLL1)
uecanL and dlscard Lhe supernaLanL and resuspend Lhe pelleL ln Lhe mannlLol medlum
1ransfer Lhe mlLochondrlal suspenslon Lo a LesL Lube and place ln an lcewaLer baLh
ulfferenLlal cenLrlfugaLlon
W 1he mosL wldely used meLhod for cellular
componenLs fracLlonaLlon
W ls used for Lhe flrsL sLeps ln cell fracLlonaLlon
because lL rapldly separaLes large membrane
organelles from smaller one
W lL ls based on dlfferences ln speed aL whlch
organelles sedlmenL Lo Lhe boLLom of a cenLrlfuge
Lube 1he selecLlon of veloclLy and duraLlon of
Llme depends on dlfferenL slze welghL denslLy
and shape of sLudylng organelles
IeloclLy sedlmenLaLlon
W SedlmenLaLlon veloclLy (so called zone
cenLrlfugaLlon) has Lhe advanLage of low speed
cenLrlfugaLlon and shorL Llme buL resulLs ln
lncompleLe separaLlon
W Lach parLlcle has Lermlnal veloclLy when
sub[ecLed Lo a cenLrlfugal fleld lL ls deLermlned
by above menLloned parLlcle characLerlsLlcs as
well as by Lhe vlscoslLy of Lhe medlum Lhey have
Lo pass Lhrough and Lhe relaLlve cenLrlfugal force
(8Cl)
SeparaLlon on Lhe basls of dlfferenL
slze and shape
W 1he larger parLlcles wlll sedlmenL fasLer Lhan
Lhe smaller one
W lrom Lhe LoLal homogenaLe we can obLaln
dlfferenL cell fracLlons (known as pelleLs)
followlng several sLeps of cenLrlfugaLlon wlLh
approprlaLe veloclLy and duraLlon of Llme
llrsL fracLlon
nuclel lnLacL cells and cell debrls
Second fracLlon
1hree dlfferenL subcellular componenLs
mlLochondrla lysosomes and peroxlsomes
IkAC1ICNS 8ASLD CN 1nL LkLkIMLN1
;uLS1lCnS
WhaL ls Lhe purpose of uslng precooled
maLerlals durlng Lhe preparaLlon of Lhe
speclmen for cenLrlfugaLlon?
1he maLerlals should be precooled ln order Lo preserve
Lhe enzymes and oLher consLlLuenLs afLer Lhey are
released from Lhe sLablllzlng forces of Lhe cell
C Mlcroscoplc LxamlnaLlon
M|croscop|c Lxam|nat|on of Nuc|ear
Iract|on
8emove a Llny amounL of Lhe nuc|ear pe||et wlLh a
spatu|a
8emove a Llny amounL of Lhe nuc|ear pe||et wlLh a
spatu|a
Smear Lhe maLerlal on clean s||de Smear Lhe maLerlal on clean s||de
lmmedlaLely add several drops of Lhe nuc|ear
sta|n |actoacetoorce|n
lmmedlaLely add several drops of Lhe nuc|ear
sta|n |actoacetoorce|n
AfLer m|ns add a covers||p and very genLly press ouL Lhe
excess sLaln wlLh a paper towe|
AfLer m|ns add a covers||p and very genLly press ouL Lhe
excess sLaln wlLh a paper towe|
Lxamlne Lhe preparaLlon under nC wlLh a green f||ter ln
place
Lxamlne Lhe preparaLlon under nC wlLh a green f||ter ln
place
When you have locaLed a reglon wlLh nuclel
swlLch Lo Lhe CIC
When you have locaLed a reglon wlLh nuclel
swlLch Lo Lhe CIC
lor each of Lhe flve round nuclel measure Lhe nuc|ear
d|ameter CeL Lhe mean
lor each of Lhe flve round nuclel measure Lhe nuc|ear
d|ameter CeL Lhe mean
NUCLLI under nC
NUCLLI under CIC
Mlcroscoplc LxamlnaLlon of
MlLochondrlal lracLlon
W rocedure
W 8esulLs and analysls
Mlcroscoplc LxamlnaLlon of
MlLochondrlal lracLlon rocedure
Obsetve ooJet nlO ooJ
OlO (temove qteeo flltet
meosote tbe slze)
koom tempetotote
fot 10 mlootes
Mlcroscoplc LxamlnaLlon of MlLochondrlal
lracLlon Culde ;uesLlon 2
W Morphology of mlLochondrla under elecLron
and llghL mlcrograph
Mlcroscoplc LxamlnaLlon of MlLochondrlal
lracLlon Culde ;uesLlon 3
W WhaL ls Lhe funcLlon of Lhe !anus
green sLaln ln mlLochondrla
sLalnlng?
8educed colorless (leucobase)
Cxldlzes bluegreen
MlLochondrla ablllLy Lo
reoxldlzed Lhe sLaln Lhus
appear bluegreen under llghL
mlcroscope
Mlcroscoplc LxamlnaLlon of MlLochondrlal
lracLlon Culde ;uesLlon 4
W Comparlson of Lhe slzes of nucleus and
mlLochondrla
Component 1r|a| 1r|a| 2 1r|a| 3 Mean
nucleus 3 m
MlLochondrla 2 m
8eference
W SeparaLlon of Sub Cellular ComponenLs 8eLrleved 4 AugusL 20 from
hLLp//wwwscrlbdcom/doc/602404/LxperlmenL2SeparaLlonofSubCellularComponenLs
W Ceneral 8lology Cells and Molecules 8eLrleved 4 AugusL 20 from
hLLp//academlcevergreenedu/currlcular/generalblologycells/Summer2003llles/MlLochondrla_lso
laLlonpdfChapLer 3 Cell lracLlonaLlon lnLroducLlon 8eLrleved on AugusL 3 20 from
hLLp//homepagesgacedu/cellab/chpLs/chpL3/lnLro3hLml
W LxperlmenL 3 uehydrogenase Lnzyme of Caullflower MlLochondrla 8eLrleved on AugusL 3 20
from hLLp//wwwcolbyedu/chemlsLry/8C368/laboraLory/LxperlmenL203pdf
W Ceneral 8lology Cells and Molecules 8eLrleved on AugusL 3 20 from
hLLp//academlcevergreenedu/currlcular/generalblologycells/Summer2003llles/MlLochondrla_lso
laLlonpdf
W lsolaLlon of MlLochondrla lrom Caullflower uslng ulfferenLlal lracLlonaLlon 8eLrleved on AugusL
3 20 from hLLp//wwwsallsburyedu/wac/excellence/elllson/elllsonhLm
W !anus Creen 8 sc203740 8eLrleved on AugusL 3 20 from hLLp//wwwscbLcom/daLasheeL
203740[anusgreenbhLml
W !ournal of PlsLochemlsLry and CyLochemlsLry 8eLrleved on AugusL 3 20 from
hLLp//[hcsagepubcom/conLenL//4/234fullpdf
W urlflcaLlon by ulfferenLlal CenLrlfugaLlon 8eLrleved on AugusL 3 20 from
hLLp//faculLyplaLLsburghedu/donaldsllsh/ulffCenLhLml
W SLudles on Lhe Mechanlsm of !anus Creen 8 SLalnlng of MlLochondrla 8eLrleved on AugusL 3
20 from hLLp//onllnellbrarywlleycom/dol/0002/ar0938007/absLracL