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