Hielscher faka'ata tekinolosia

Ultrasonic teuteu ʻa C. Elegans sipinga

C. elegans, ko ha ʻuanga nematode, ko ha sipinga ia ʻoku lahi hono fakaʻaongaʻi ʻi he paiolosii. ʻOku fie maʻu ʻa e sipinga ʻo e teuteu kimuʻa pea toki ʻanalaiso lysis, ivi mo e toʻo ʻuli pea pehe ki he RNA fragmentation, ʻa ia ʻe lava ke reliably fakahoko ʻo fakafou ʻi he sonication. Ko e ultrasonic toʻotoʻo disruptors ʻoku falalaʻanga, lelei mo faingofua ke fakaʻaongaʻi ki he teuteu ʻaukai ʻa c. elegans sipinga.

Ultrasonic teuteu ʻa C. Elegans sipinga

C. elegans ʻoku roundworms, ʻa ia ʻoku fakaʻaongaʻi lahi ʻi he fekumi laboratories ke fakatotoloʻi ʻa e genomics, paiolosii, mo e ngaahi mahaki fakalakalaka. ʻOku tokolahi ha afa ʻi he genome ʻo C. elegans ʻoku ʻi ai honau kaunga ngaue ʻi he kau tau. Ko ia ai, ko e ʻuanga nematode ko ha sipinga ʻaonga ʻaupito ia ki he ngaahi mahaki ʻo e tangata. Ko e ngaahi lelei kehe ki hono fakaʻaongaʻi lahi ʻo e C. elegans ko hono tauhi faingofua mo fakama ia ʻi he ngaahi peleti ʻoku ʻi ai e pekitilia (e.g., E. kololi), ʻene femahinoʻaki, ngaue lelei, pea pehe ki he malava ke maʻu e momoko mo tauhi e fanga ʻuanga ʻi ha vahaʻataimi loloa ange.
Ko e ivi mo hono ʻanalaiso ʻo e ngaahi meʻa ʻoku fai ki ai e laboratories mo ultrasonic sipinga ʻo e teuteu ko e founga ia kuo fokotuʻu ke lyse C. elegans nematodes ʻi he tuʻunga fakalakalaka kotoa pe (hange ko e embryos, sivi L1-L4, kakai lalahi). Koeʻuhi ʻoku toe fakaʻaongaʻi foki ʻa e elegans C. ko ha founga ke fakahaaʻi ʻaki ʻa e polotini kuo ʻosi fakapapauʻi, ko ha founga falalaʻanga, reproducible lysis mo e ivi toʻo ʻuli, ʻa ia ʻoku ne ʻomi ha ivi lahi, ʻoku fie maʻu. ʻOku lava ke maʻu ʻa e ultrasonic toʻotoʻo mo e founga toʻo ʻuli ko e fekumi-type homogenizers pea mo ha sipinga fakavahaʻa-ultrasonicators. Ko e teuteu ki he sipinga ʻo e teuteu mo e faʻahinga sipinga kotoa pe ,, Hielscher Ultrasonics maʻu ʻa e ultrasonic toʻotoʻo lelei taha ki hoʻo founga ngaue ki he loki fakatotolo.
C. elegans is a widely used model organism in biological research. Ultrasonic lysis is a sophisticated, reliable, reproducible and rapid technique to prepare high-quality protein extracts from C. elegans.

Ultrasonic C. elegans Lysis ki he

  • Teuteuʻi ʻo e ʻuanga homogenates
  • Polotiini to'o e 'uli
  • Toʻo ʻaki e tatau fakamoimoú ʻa e ʻu
  • Ivi ʻoku ne fakamatalaʻi
  • Immunoprecipitation
  • Fakahihifo 'o blotting
  • Toʻo e ʻulí mei he RNA
  • ʻOku pehe ʻe Enzymatic as
ʻOku ʻi ai ha ultrasonic ʻe valu ʻi he sonotrode MTP-24-8-96 probes ki he sonication ʻo e ngaahi peleti microtiter.

ʻOku ʻi ai ha ultrasonic ʻe valu ʻi he sonotrode MTP-24-8-96 probes ki he sonication ʻo e ngaahi peleti microtiter.

Kole 'a e fakamatala




Fakatokanga'i ange 'etau Tu'utu'uni totonu fakatautaha.


Ultrasonic ki he C. Elegans fakahohaʻasi mo e Lysis

ʻE lava ke fakahoko ʻa e ultrasonic homogenization mo e lysis ʻo C. elegans pea mo e ivi mo e toʻo ʻuli ʻi hono fakaʻaongaʻi ʻo e ngaahi founga kehekehe ʻo fakaʻaongaʻi ʻa e homogenization mo e lysis buffers etc. ʻOku faitatau ʻa e lysis kotoa pe mo e ngaahi sipinga kuo pau ke tauhi hokohoko ʻi he ʻaisi ke taʻofi ʻa e ivi maʻulalo. ʻI lalo, ʻoku mau ʻoatu ai ha ngaahi ultrasonic falalaʻanga mo vave lysis mo e toʻo ʻuli ki hono teuteuʻi ʻo ha ivi ʻoku maʻolunga pe ko e ngaahi sipinga ʻoku ʻi ai ʻa c. elegans sipinga.

Ngaahi lelei ʻo ultrasonic C. elegans Lysis

  • falala'anga
  • ngaahi
  • fakapatonu pe hono puleʻi
  • mapuleʻi ʻo e founga falalaʻanga
  • founga angavaivai
  • faingofua hono fakaʻaongaʻí
  • malu

Ultrasonic ivi toʻo ʻuli mei he C. elegans sipinga

ʻE lava ke fakahoko ʻa e ultrasonic lysis mo e ivi toʻo ʻuli ʻo e C. elegans fanga ʻuanga ʻaki hano fakaʻaongaʻi ha ngaahi founga kehekehe. ʻOku mau ʻoatu ʻi lalo ha ngaahi lysis falalaʻanga mo vave ki he ngaahi ola reproducible hono toʻo e ʻuli.

Teuteu vave ʻo e fakaʻofoʻofa toʻo mei he C. elegans fanga ʻuanga ʻe Sonication

ʻI he ngaahi founga ko ʻeni te ke lava ʻo teuteuʻi ʻa e C. elegans lysates ʻi ha miniti ʻe 30.
Fakatahatahaʻi ʻo e C. elegans
Fili ʻa e elegans ʻoku fie maʻu ki ha foʻi tiupi 1.5 ml phosphate buffered Saline (PBS) pe fufulu kinautolu mei ha peleti mo 1.5 ml PBS. Centrifuge ʻi ha miniti ʻe 1ʻi he 2000rpm ki he pellet. Tauhi ʻa e ngaahi sipinga ʻi he taimi kotoa pe ʻi he ʻaisi.
Hili ia, fufulu tuʻo ua ʻa e fanga ʻuanga ʻaki ʻa e PBS.
Hili ia, fufulu tuʻo ua ʻa e fanga ʻuanga ʻaki ʻa e ddH2O.
Resuspend ʻa e fanga ʻuanga ʻi he 500ul ʻo homogenization ngataʻanga (HB). ʻOku mateuteu he taimi ni ʻa e ngaahi sipinga ʻuanga ki ultrasonic lysis.
Ke maʻu ha ivi ʻoku maʻolunga ange, mahalo te ke fie fakasiʻisiʻi hangatamaki fakaʻuliʻi ʻaki hono fufulu e fanga ʻuanga ki 5miniti ʻe taha ʻi he PBS mo e meʻa ʻoku ʻikai ke lelei, ultra vai maʻa (ddH2O) pe fakahoko sucrose floatation. Tauhi maʻu pe ʻa e ngaahi sipinga ʻuanga ʻi he ʻaisi.

Ultrasonic C. elegans Lysis Protocol

  • Fakapapauʻi ʻoku ke teuteuʻi ʻa e ultrasonicator upfront koeʻuhi ke mateuteu ʻa e ultrasonic homogenizer ke fakaʻaongaʻi (fekumi kaka hake, polokalama sonication kimuʻa).
  • Ultrasonicator UP200Ht (200 watts, 26kHz) with microtip S26d2 for the preparation of biological samplesKapau ʻoku tuʻu maʻu hoʻo ultrasonicator, fokotuʻu ʻa e ʻaisi ʻaki hoʻo sipinga ʻo e tiupi ʻi lalo ʻi he ultrasonic fekumi pea fakahu ʻa e ultrasonic fekumi ki he foʻi tiupi 1.5 ml.

  • Kia C. elegans lysis mo e UP200St pe UP200Ht, ultrasonication ʻoku totonu ke fakahoko ia ʻaki hano fakaʻaongaʻi ʻo ha microtip (e.g., 2mm fekumi S26d2; vakai ki he fakatata toʻohema) ʻi he 40% amplitude ki 1sec mo 30sec longo ʻi he vahaʻa ʻo e. 5 sonication siakale ki he 1 takitaha ʻoku ʻi ai 30sec fanga kiʻi longo ʻoku feʻunga mo c. elegans lysis. Kapau te ke fuofua fakahoko ʻa e lysis, te ke lava ʻo vakaiʻi ʻa e fakalakalaka lysis ʻi he fanga kiʻi aliquots iiki ʻo e sipinga hili hono ta takitaha ʻo fakaʻaongaʻi ha meʻa-fakaʻata.
  • ʻOku ola lelei hono fakakakato ʻo e Lysis ʻi he taimi ʻoku motuhia ai ʻa e fanga ʻuanga. ʻOku ʻi ai ha ngaahi ola sonication lahi ʻi he breakage ʻo nuclei pea ʻoku ʻasi ia ʻi he taimi ʻoku viscous pe foams ai ʻa e sipinga. Ke taʻofi ha sipinga maʻulalo, fakaʻaongaʻi ha pulses lahi ange ʻo ka fie maʻu. ʻOua naʻa ke fakalahi ʻa e taimi ʻo e siakale ultrasonic kotoa pe ke maʻu ai ha ivi lelei.
  • Fakaʻataʻata ʻa e lysate toʻotoʻo ʻaki centrifuging ʻa e ultrasonically lysed fanga ʻuanga ʻi he 14, 000rpm ki 10miniti ʻe 4ºC.
  • Hili ia, ʻave ʻa e supernatant ki ha foʻi tiupi foʻou pea teuteu ki he immunoprecipitation pe ngaahi lea kehe.

Fakatokangaʻi ange ki he homogenization ngataʻanga: teuteuʻi ʻa e homogenization ngataʻanga ki he ultrasonic lysis Protocol ʻi ʻolunga ʻo hange ko ʻeni:

  • 15 mM Hepes pH 7.6 – 15 ml ʻo e 0.5 M
  • 10 mM KFl – 2.5 ml ʻo e 2 M
  • 1.5 mM MgCl2 – 0.75 ml ʻo e 1 M
  • 0.1 mM EDTA – 100 ul ʻo e 0.5 M
  • 0.5 mM EGTA – 2.5 ml ʻo e 0.1 M
  • 44 MM Sucrose – 14.7 ml ʻo e 50%
  • Tanaki atu ki muʻa pea toki fakaʻaongaʻi: 1mM DTT – 1000x ʻo 1M
  • kau ai mo ha protease inhibitor

Ultrasonic Lysis ʻo C. elegans ki quantitative fakamaʻa ʻo e ʻofa

C. elegans embryos (∼2 million ki he ako) naʻe toki utu pe ia ʻi he triplicate totonu ʻe bleaching kei talavou hermaphrodites mo sonicated ʻi he ʻaisi (siakale: 0.5 s, amplitude: 40 – 45%, 5 kakau/fakataha, fakataha ʻe 5, ʻi he vahaʻa ʻo e ngaahi fakatahaʻanga: 30; UP200S ultrasonic processor mo e S26d2 Micro (Hielscher Ultrasonics GmbH)) ʻi lysis ngataʻanga (voliume fakakatoa: ∼600 μl; 50 MM Tris-HCl, pH 7.4, 100 MM KFl, 1 MM MgCl2, 1 MM EGTA, 1 MM DTT, 10% glycerol, protease inhibitor fefiofi, 0.1% Nonidet P-40 fetongi). Hili sonication, nonidet P-40 naʻe tanaki atu ʻa e fetongi ki he 1% pea naʻe incubated ʻa e lysates ʻaki ʻa e vilo ʻi he 4 ° c ʻi ha miniti ʻe 30, hoko ai mo centrifugation ʻi he 20,000 × g ʻi ha miniti ʻe 20 ʻi he 4 ° C. Naʻe fakaʻata leva ʻa e lysate ʻo ʻikai toe fakahohaʻasi ʻa e foʻi kuta ʻi ʻolunga pea mavaeua ʻa e vaeua ʻo e kau ʻAnitai-GFP agarose pe naʻe ʻasi ʻa e control ʻoku ne puleʻi (40 – 50 μl). Hili e vilo ʻa e hiku ʻi he 4 ° c ʻi ha miniti ʻe 60 – 90, naʻe fufulu tuʻo taha pe ʻa e ʻasi ʻi lysis ngataʻanga ʻoku ʻi ai ʻa e 0.1% Nonidet P-40 fetongi, hoko ai mo ha tuʻo ua ʻo e foo (25 MM Tris-HCl, pH 7.4, 300 MM Akacl, 1 MM MgCl2) pe ngataʻanga ʻo e II (1 MM Tris-HCl, pH 7.4, 150 MM NaCl, 1 MM MgCl2) pe fakatouʻosi. Ki he GFP: MBK-2, naʻe fakahoko ha fakatotolo kehekehe ʻe ua ʻo fakaʻaongaʻi ki ai ha ngaahi tuʻunga fo kehekehe. Naʻe holoki orbital ʻe orbital tetetete ʻi he 50 μl ʻo e 6 m urea/2 M thiourea ʻi he mafana ʻo e loki. Ki he MBK-1:: GFP ngaahi fakatotolo ʻahiʻahi, polotini naʻe liunga ua hono luluʻi ʻi he 50μl ʻo e 8 m guanidinium chloride ʻi he 90 °C, hoko ai mo ethanol precipitation. Naʻe ha leva ʻa e ngaahi sipinga ʻo e ivi ʻi he founga fakaleleiʻi.
(FF. Seni et Al., 2016)

VialTweeter at the ultrasonic processor UP200ST

VialTweeter sipinga ʻo e ʻiuniti teuteu fakavahaʻa-taʻu ki he hoko sonication ʻo ha tiupi sivi ʻe 10.

Ultrasonic ʻUanga Homogenization mo Lysis

Ki he C. elegans lysis mo e ivi toʻo ʻuli, 30,000 nemotodes ʻo e tuʻunga mahuʻinga naʻe tanaki ki he sipinga mo e fo ʻi he ʻaisi-momoko S-basal, ʻa ia naʻe fakatefito ʻi he centrifugation ʻi he 1500 rpm ʻi ha miniti ʻe 2., pea toki fufulu tuʻo ono ia ʻaki ʻa e ʻaisi-momoko ke toʻo ʻa e pekitilia, pea toki tanaki ʻi he ʻaisi Ke maʻu ha ivi ke toʻo ʻaki e ʻuli, naʻe fakangofua ke faʻu ʻe he ngaahi ʻunufe ʻa e kakai lalahi ke nau faʻu ha pellet ʻi he ʻosi ʻa e fo fakaʻosi ʻo e S. Naʻe toe nofoʻi leva ʻa e pellets ʻuanga ʻi he 1 ml ʻo e toʻo ʻuli ʻi he ʻaisi [20 mM e sotiume phosphate, pH 7.4, 2m mahalo EDTA, 1% Triton-X-100, protease inhibitors (Sigma P2714)] mo e ngaue he taimi pe ko ia.
∼30,000 gravid-ʻunufe (fekauʻaki mo e ∼100 mg viviku), naʻe sonicated ʻi he ʻaisi ʻo fakaʻaongaʻi ha faʻahinga fekumi ultrasonicator (e.g. UP50H mo microtip MS2) ʻi he 40% amplitude ki he siakale ʻe 10 ʻo e 3 sec ʻi he ʻaisi, 30 sec (FF. Baskharan et Al. 2012)

Ultrasonic misini toʻo ʻuli mei he C. elegans

ʻI lipidomics, ko ha kolo ʻo e metabolomics, ʻoku fakafotunga mo analysed ai ʻa e ngaahi founga totonu. C. elegans ʻoku lahi hono fakaʻaongaʻi ʻi he lipidomics ke vakaiʻi ʻa e fengaueʻaki ʻa metabolic lipids mo honau ngaahi nunuʻa ʻi he moʻui lelei mo e lifespan.
ʻOku fakaʻaongaʻi ʻa e ultrasonic lysis mo e toʻo ʻuli ke tukuange lipids hange ko e sphingolipids mei he C. elegans embryos, sivi, mo e ngaahi ʻunufe lalahi. ʻOku fakaʻaongaʻi ʻa e Ultrasonication ke teuteuʻi ʻaki ʻa e ʻuanga homogenates pea toki toʻo ʻa e lipids mei he sipinga.
Protocol maʻa e ultrasonic misini toʻo ʻuli mei he C. elegans
ʻAisi kovi ʻene e C. elegans pellet ʻi he ʻaisi pea resuspend mo e 0.5 ml ultrawater. 
Sonicate ʻa e c. elegans sipinga ʻi he 1, 5mL siviʻi e tiupi, lolotonga ia ʻoku hokohoko atu hono tauhi e ngaahi sipinga ʻi he ʻaisi.
ʻE lava ke fakahoko ʻa e Sonication ʻo fakaʻaongaʻi ha fekumi-ultrasonicstor hange ko e UP200Ht, ʻa e sipinga ultrasonic teuteuʻi ʻo e ʻiuniti VialTweeter (hoko sonication ʻo ha sipinga ʻe 10) pe ko e UIP400MTP (ki he sonication ʻo e ngaahi peleti fakavahaʻa-taʻu hange ko e ʻu lauʻi peleti lelei ʻe 96). ki ultrasonic lysis mo e UP200Ht fakaʻaongaʻi ʻa e tokoni Micro S26d2. tomuʻa fokotuʻu ʻa e founga ʻo e siakale ultrasonic ʻi he digital menu. Fokotuʻu ʻa e amplitude ki he 10% mo sonication cycle Mode ʻo e 2 Sec pulses ʻo e siakale ʻe 20 mo e kiʻi malolo ʻi he 30 Sec. ʻi he vahaʻa ʻo e ultrasonic pulsation takitaha.
ʻAve ʻa e supernatants ki he tiupi sioʻata ʻaki ʻa e tata sikalu. 
Fakahoko Folch toʻo ʻuli ʻaki hono tanaki atu e 1 ml ultrawater ki he foʻi leta sioʻata takitaha, hoko atu ai mo hono tanaki atu e 6 ml ʻo e chloroform/methanol (fika ( ratio) ki he foʻi leta sioʻata takitaha.
Vortex e foʻi leta sioʻata takitaha ki he Sec ʻe 30 ʻi ha tuʻo 4. 
Centrifuge ʻa e tiupi ʻi he 1,258 x g ʻi ha miniti ʻe 15 (Eppendorf, 5810 R) ke fakalahi atu ki he konga ʻo e mavahevahe. 
ʻAve ʻa e konga siʻi hydrophobic ki ha foʻi tiupi sioʻata maʻa ʻaki ha ipu sioʻata Pasteur pipette. 
Fakamomoa ʻa e konga siʻi hydrophobic ʻi lalo nitrogen tafe mai ʻi ha nitrogen evaporator. 
Tauhi ʻa e pellet momoa ʻi ha-80 ° c ʻaisi kae ʻoua kuo fakaʻaongaʻi.

Ultrasonic teuteu ʻo e ʻUanga Lysates

ʻUanga lysate: L4 naʻe toli mo fufulu tuʻo tolu ʻa e ngaahi ʻunufe ʻi he funga siteisi ʻaki ʻa e M9 ngataʻanga (42.26 mM Na2HPO4, 22.04 mM KH2PO4, 85.56 mM Akafl, mo e 0.87 mM MgSO4) kae lava ke toʻo kotoa ʻa e pekitilia. Hili hono toʻo e lahi taha ʻo e ngaahi M9, naʻe resuspended ʻa e fanga ʻuanga ʻi lysis ngataʻanga: 50 mM HEPES, 50 mM KFL, 1mM EDTA, 1mM EGTA, 5 mM phosphate β-glycerol, 0.1% (v/v) Triton X-100, 50 mM ʻelemeniti ko fluoride, 1 mM ʻelemeniti ko orthovanadate, 5 mM ʻelemeniti ko pyrophosphate, 0.2 mM phenylmethanesulylfluoride mo e protease inhibitor. Naʻe momoko ʻa e fanga ʻuanga ʻi he nitrogen mo e thawed ʻi he 37 ° c ʻi ha tuʻo tolu, pea naʻe sonicated ʻa e fanga ʻuanga ʻi he ʻaisi momoa ʻaki ha ʻiuniti ultrasonic VialTweeter ki hono teuteuʻi hoko ʻo ha tiupi ʻe 10. Naʻe fakahoko ʻa e Sonication ʻi he 50% amplitude ʻi ha siakale ʻe 10 ʻo e 2 Sec. mo e 30 Sec kiʻi malolo ʻi he vahaʻa ʻo e sonication. Hili ia, naʻe centrifuged ʻa e ngaahi sipinga ʻi he 12000 rpm ʻi he 4 ° c ʻi ha miniti ʻe 15. Naʻe tanaki mo tauhi ʻa e supernatant ʻi he 70 ° c. Naʻe fakaʻaongaʻi ha aliquot ki ha ivi feʻunga naʻe fai ʻe Peletifooti.
Fakapapauʻi ʻo e glutathione fakakatoa, GSH, mo e GSSG: ki he glutathione, naʻe fai ʻa e lysates mo e loto fakapapau ʻi he ʻaho tatau pe. Suka-fafangaʻi mo mapuleʻi L4 sivi naʻe utu pea fufulu ʻaki e M9 tuʻo tolu. Hili hono toʻo e lahi taha ʻo e ngaahi M9, naʻe resuspend ʻa e fanga ʻuanga ʻi he ʻaisi metaphosphoric ʻesiti (5% w/v), pea naʻe sonicated ʻa e fanga ʻuanga ʻi he ʻaisi ʻaki ha ultrasonic VialTweeter ʻi he 50% amplitude ʻi he siakale ʻe hongofulu sonication ʻo e 2 Sec. Hili ia, centrifuged ʻi he 12000 rpm ʻi he 4 ̊C ʻi ha miniti ʻe 15.
(FF. Alcántar-Fernández et Al., 2018)

C. elegans sipinga kimuʻa pea toki teuteuʻi Immunoprecipitation mo e fakahihifo ʻo e Hihifo

Ko hono fakanounou, ko e ngaahi meʻa embryonic, C. elegans L1 sivi naʻe tupu ia ʻi ha ngaahi anga fakafonua lahi ki he kakai lalahi. Naʻe tanaki ʻa e Embryos ʻo fakaʻaongaʻi ʻa e founga holoki angamaheni mo e tuku ʻi lysis ngataʻanga (50 mM Tris, pH 7.5, 100 mM KFl, 1 mM EDTA, 1 mM MgCl2, 8.7% glycerol, 0.05% NP-40, 1􏰅 Protease Inhibitor, mo e 1􏰅 Phosphatase Inhibitor mo e II), ʻo vave ʻene ngaue ʻaki ʻa e ultrasator UP200Ht mo e S26d2 ki he pulses ʻe 10 tupu ʻi he 10% amplitude. Kapau kuo pau ke teuteuʻi ha ngaahi sipinga lahi ange ʻi he ultrasonic VialTweeter pe ʻoku fokotuʻu atu ʻa e MultiSample-Ultrasonicator UIP400MTP ki he ʻu lauʻi peleti lelei. Hili sonication, naʻe tomuʻa fakaʻata ʻa e ngaahi fakamatala ʻe centrifugation ʻi he 30, 000g ʻi ha miniti ʻe 20 ʻi he 4 ° c. Naʻe incubated ʻa e konga naʻe toʻo kimuʻa (300μg ʻo e ivi fakakatoa) ʻaki ʻa e 40μ􏰂g ʻo e kau ʻAnitai-FDC-25.1 antibody (ako ko ʻeni) ʻoku fakafehokotaki ki he kolosi ke maʻu ha ivi a-agarose, pe ko hono puleʻi, ko ha kiʻi lapisi tatau immunoglobulin (Ig) G naʻe fakaʻaongaʻi ia ki he ivi ʻo e 200μl ʻo lysis ngataʻanga ʻoku ʻi ai ʻa e 1% NP-40, ʻa ia ʻoku ne fakasiʻisiʻi ʻa e ʻikai ke ʻi ai ha polotini pau ki he matrix. Naʻe fetongitongi ʻa e ngaahi sipinga ko ʻeni ʻi he 1 h ʻi he 4 ° c, naʻe fufulu tuʻo tolu ʻa e ʻasi ʻi he lysis ngataʻanga, pea holoki ʻaki ʻa e 30μl ʻo e glycine/HCl mo e 200 mM Akafl, pH 2.2. Hili immunoprecipitation, naʻe holoki ʻa e eluates ʻi he 30μ􏰂l ʻo e sipinga ʻo e ngaahi meʻa ne fuʻu lahi, ʻo fakamafanaʻi ki he 95 ° c ʻi ha miniti ʻe 4, pea ko e 3% ʻo e fakakatoa ki he input mo e 30% ki he eluates naʻe fakaʻaongaʻi ia ki he peesi ʻo e SDS-hihifo pea hoko atu ai mo e kau ʻAnitai-FDC-25.1 (1:400), ʻAnitai-LIN-23 ((1:750), ʻAnitai-ubiquitin (1:1000), Anti-GSK3􏰁 (1:500), pe anti-􏰁β-Actin (1:2000) maluEi. ʻI he feituʻu naʻe ʻikai ke immunoprecipitation ai ʻa e ngaahi meʻa naʻe toʻo, naʻe toe fakaava ʻa e lahi fakakatoa ʻo e ivi mei he ngaahi meʻa ko ia ʻi he sipinga ʻo e ngaahi meʻa ko ia, ʻo fakamafanaʻi ki he 95 °C, pea toki fakaʻaongaʻi fakahangatonu ki he peesi ʻo e Blotting Hihifo. (FF. Segref et Al. 2020)

Vakili-fa'ahinga insonifier UP200St ki he lysis

Ultrasonic toʻotoʻo disruptor UP200St mo e S26d2 Micro ki he lysis mo e ivi toʻo hingoa

Ultrasonic Lysis ʻi he malumalu ʻo e mafana ʻo e ʻea

The VialTweeter is a MultiSample Ultraonicator that allows for reliable sample preparation under precisely controlled temperature conditions.ʻOku mahuʻinga ʻa e mapuleʻi totonu mo falalaʻanga ʻo e ʻea ʻi he taimi ʻoku nau tokangaʻi ai ʻa e ngaahi sipinga totonu. ʻOku kamata ʻe he mafana ʻo e ʻea ʻa e thermally ʻoku ne fakaʻaiʻai ʻa e maʻulalo ʻi ha ngaahi sipinga.
ʻI he hoko ʻa e sipinga fakamisini kotoa pe, ʻoku fakatupu ʻe sonication ha vela. Neongo ia, ʻe lava ke mapuleʻi lelei e mafana ʻo e ngaahi sipinga ʻi he taimi ʻoku fakaʻaongaʻi ai e VialTweeter. ʻOku mau ʻoatu ha ngaahi fili kehekehe ke vakaiʻi mo puleʻi ʻaki e mafana hoʻo sipinga lolotonga hono teuteuʻi kinautolu ʻaki e VialTweeter mo e VialPress ke ʻanalaiso.

  1. Vakaiʻi ʻa e sipinga ʻo e ʻea: ko e ultrasonic processor UP200St, ʻa ia ʻoku ne teke ʻa e VialTweeter, ʻoku fakanaunau ʻaki ia ha polokalama fakakomipiuta fakapotopoto mo ha mafana pluggable sensor resistance. Palaki ʻa e mafana ʻo e ʻea sensor resistance ki he UP200St pea fakahu ʻa e tokoni ʻa e ʻea sensor resistance ʻi he taha ʻo e ngaahi sipinga ʻo e tiupi. ʻI he naunau fakaʻilekitulonika coloured, te ke lava ʻo fokotuʻu ʻi he menu ʻo e UP200St ha mafana pau ki hoʻo sipinga sonication. ʻE taʻofi leva ʻa e ultrasonicator ʻi he taimi ʻe aʻu ai ʻa e mafana ʻo e ʻea pea kiʻi malolo kae ʻoua kuo aʻu ʻa e sipinga ʻo e ʻea ki he tuʻunga ʻulungaanga mahuʻinga maʻulalo ange ʻo e mafana ʻo e ʻ∆ mafana. Pea toe kamata leva ʻa e sonication. ʻOku taʻofi ʻe he founga fakapotopoto ko ʻeni ʻa e maʻulalo ʻoku ne fakaʻaiʻai ʻa e ʻea.
  2. ʻE lava ke tomuʻa ʻita ʻa e konga VialTweeter. Faʻo ʻa e konga VialTweeter (ko e sonotrode pe taʻe transducer!) ki he ʻaisi pe ʻaisi ke teuteu ke mokomoko ʻa e konga titanium ʻoku tokoni ke toloi ʻa e mafana ʻo e ʻea ʻi he sipinga. Kapau ʻe lava, ʻe lava ke tomuʻa ʻita foki mo e sipinga.
  3. Fakaʻaongaʻi ʻa e ʻaisi momoa ke mokomoko lolotonga e sonication. Fakaʻaongaʻi ha laulau mamaha ʻoku fonu ʻi he ʻaisi momoa pea tuku ʻa e VialTweeter ʻi he ʻaisi momoa ke lava ʻo molia vave ʻa e vela.

Kumi ʻa e tokolahi lelei ultrasonic toʻotoʻo Disruptor ki hoʻo tohi kole Lysis

Ko Hielscher Ultrasonics ko ha kautaha taukei taimi loloa ia ʻo e ngaue maʻolunga ultrasonic disrupters mo homogenizers ki laboratories, ngaahi founga meʻafua ki ʻolunga mo e ngaahi meʻa fakamisini. Ko hoʻo hangatamaki lahi ʻo e anga fakafonua, ko hoʻo fakatotolo pe taumuʻa ʻo e ngaue pea mo e lahi ʻo e meʻa toʻotoʻo ke fai ʻi he houa pe ʻaho, ko ha ngaahi meʻa mahuʻinga ia ke maʻu ai ʻa e ultrasonic toʻotoʻo disruptor ki hoʻo tohi kole.
ʻOku ʻomi ʻe Hielscher Ultrasonics ha ngaahi founga kehekehe ki he hoko sonication ʻo e ngaahi sipinga fakavahaʻa-taʻu (ʻo aʻu ki he ngaahi sipinga ʻe 10) pea pehe ki ha ngaahi sipinga lahi (hange ko e.e, microtiter ʻu lauʻi peleti/96-ʻu lauʻi peleti lelei), ko e fale fakatotolo ʻiloa fekumi ultrasonicator mo e ngaahi tuʻunga kehekehe ʻo e malohi mei he 50 ki he 400 Watts ki he ngaahi meʻa lalahi ultrasonic processors ʻo aʻu ki he 16,000watts ki he ʻiuniti ki he fakahohaʻasi fakakomesiale mo e ivi toʻo hingoa ʻi ha faiva lahi. ʻOku langa ʻa e Hielscher ultrasonicators kotoa pe ki he ngaue 24/7/365 ʻi he kavenga kakato. Ko e Robustness mo e ala falalaʻanga ko e ngaahi tefitoʻi fotunga ia ʻo ʻetau ngaahi meʻangaue ultrasonic.
ʻOku fakanaunau ʻa e ultrasonic fakaʻilekitulonika kotoa pe homogenizers ʻaki ʻa e polokalama fakakomipiuta poto, coloured ala ki he fakaʻaliʻali mo e fakamatala fakahangatonu protocolling, ʻa ia ʻoku ne ʻai ʻa e meʻangaue ultrasonic ki ha meʻangaue ʻoku faingamalie ke fai ʻi he fale fakatotolo fakakemi mo e ngaahi naunau ngaohiʻanga naunau.
Tau ʻilo muʻa, ko e ha ʻa e faʻahinga selo, ko e ha ʻa e voliume, mo e tuʻo lahi pea mo e meʻa ʻoku fakataumuʻa ke ke fakahoko ʻaki hoʻo ngaahi sipinga totonu. Te mau fokotuʻu atu ʻa e ultrasonic toʻotoʻo lelei taha disruptor ki hoʻo ngaahi fie maʻu ki he founga ngaue.

ʻOku ʻoatu ʻe he tepile ʻi lalo ha fakaʻilonga ʻo e fakafuofua ki he tuʻunga ʻo ʻetau ngaue ultrasonic mei he nima homogenizers mo MultiSample Ultrasonicators ki he ngaahi ultrasonic processors ki he ngaahi polokalama fakakomesiale:

Kulupu (batch) 'o e tohi 'Oku tafe mai 'a e 'ea 'Oku fokotu'u atu 'a e ngaahi me'angaue
96-ngaahi lauʻi peleti/microtiter n.a. UIP400MTP
10 à 0.5 ki he 1.5 mL n.a. VialTweeter 'i he UP200St
0.01 ki he 250mL 5 ki he 100mL/miniti UP50H
0.01 ki he 500mL 10 ki he 200mL/miniti 'e UP100H
mL 'i he 10 ki he 2000 20 ki he 400mL/miniti 'e UP200Ht, UP400St
0.1 ki he 20L 0.2 ke 4L/miniti 'e UIP2000hdT
10 ki he 100L 2 ki he 10L/miniti 'e UIP4000hdT
n.a. 10 ki he 100L/miniti 'e UIP16000
n.a. lalahi fakataha'i 'o e UIP16000

Fetu'utaki mai kiate kimautolu! / Kole kiate kitautolu!

Kole ki ha fakamatala lahi ange

Kataki 'o ngaue 'aki e foomu 'i lalo ke kole ha toe fakamatala ki he ultrasonic processors, faka'aonga'i mo e totongi. Te mau fiefia ke alea'i e founga 'oku mo 'i ai mo ho'omou ngaahi fie ma'u ultrasonic he polokalama!









Kataki 'o fakatokanga'i ange 'etau Tu'utu'uni totonu fakatautaha.


Ultrasonic high-shear homogenizers are used in lab, bench-top, pilot and industrial processing.

Hielscher Ultrasonics ʻa e ngaohiʻanga koloa maʻolunga ultrasonic homogenizers ki hono tuifio ʻo e ngaahi polokalama, fakamovetevetea, emulsification mo e toʻo hingoa ʻi he fale fakatotolo fakakemi, pailate mo e meʻafua ki he ngaahi meʻa fakapisinisi.

Ngaahi tohi/fakamo'oni fakafolofola



Ngaahi mo'oni'i me'a 'oku mahu'inga ke 'ilo'i

Caenorhabditis elegans

C. elegans ko ha nematode ʻoku ʻikai puli (roundworm) fekauʻaki mo e 1 MM loloa, ʻa ia ʻoku ne tafunaki ʻa e pekitilia (e.g. kololi) pea ʻoku ʻi ai ha siakale nounou ʻo e moʻui. ʻI he 20 °C, ʻoku ʻavalisi e mafasia ʻo e fale fakakemi ʻo C. elegans (N2) lifespan ʻi ha uike ʻe 2 – 3 mo ha taimi ʻo e toʻu tangata ʻi he ʻaho ʻe 3 ki he 4. ʻI he taimi ʻoku tupu tokolahi ai ʻa C. elegans, ʻa ia ʻe faingofua pe hono fakahoko ʻi he ngaahi tuʻunga ʻo e loki fakatotolo ʻoku puleʻi, ʻe faingofua pe ke screened ki he tefitoʻi moʻoni ʻo e faitoʻo konatapu pea pehe ki heʻenau ngaahi ola mo e fengaueʻaki ʻi he ngaahi founga faingataʻa molecular ʻi he mahaki ʻo e tangata. ʻOku hanga ʻe he kiʻi genome nounou, siakale nounou ʻo e moʻui mo e fanga kiʻi ngaue faingofua ʻi he ngaahi fale fakakemi ʻo ngaohi ʻa e C. elegans ko ha sipinga lelei ki he fekumi hange ko e genomics, proteomics, paiolosii, fakatotolo ki he mahaki, fakalakalaka ʻo e faitoʻo konatapu mo e ala meʻa pehe.
ʻE lava ke hoko ʻa Caenorhabditis elegans fanga ʻuanga ko ha tangata pe hermaphrodite. Hermaphrodites fakatou maʻu ʻe he tangata mo e fefine ha ngaahi ʻokani ʻoku ola lelei. Neongo ia, ʻoku ʻikai ke ʻi ai ha fanga ʻuanga fefine. Te Hermaphrodites lava ʻo moʻui fakafalala pe kiate kita pe ʻe lava foki ke tupu mo ha ngaahi foʻi ʻunufe tangata. C. elegans lava ke ne ʻomi ha fuaʻimoa ʻe 1,000 ʻi he ʻaho kotoa pe.
Koeʻuhi ko c. elegans ko e taha ia ʻo e organisms faingofua taha ʻoku ʻi ai ha founga tailiili, ʻoku fakaʻaongaʻi ʻa e nematode ʻuanga talu mei he 1963 ko ha sipinga ʻo e fekumi. ʻOku ʻikai ke lava ʻe he neurons ʻo fai ha ngaue vela, pea ʻoua naʻa ke fakahaaʻi ha ngaahi senolo voltage gated ʻelemeniti ko. ʻI he hermaphrodite, ko e polokalama ni comprises 302 neuron ʻa e sipinga kuo ʻosi fikaʻi, ʻi he meʻa ʻoku ʻiloa ko ha connectome.
Ko e lahi taha ʻo e ngaahi afa ʻi he C. elegans genome ʻoku ʻi ai honau kaunga ngaue lelei ʻi he tangata ʻa ia ʻoku ne ʻai ke hoko ia ko ha sipinga ʻaonga ʻaupito ki he ngaahi mahaki ʻo e tangata pea ʻoku fakaʻaongaʻi ia ke ako ai ʻa e paiolosii, ageing mo e ngaahi moʻoniʻi meʻa ʻoku ne takiekina longevity. ʻIkai ngata ai, C. elegans mutants ʻomi ha sipinga ki he ngaahi mahaki lahi ʻo e tangata kau ai ʻa e moveuveu neurological (e.g. Alzheimer), fakahoko mahaki mafu mo e mahaki kofuua.
Naʻe ngaohi ʻe he ngaahi moʻoniʻi meʻa ko ʻeni ʻa C. elegans ha sipinga mahuʻinga ʻaupito ki ha ngaahi feituʻu fekumi lahi. Ko ia ai, C. elegans ko e fuofua meʻa multicellular ia ke maʻu kotoa hono genome sequenced. ʻOku ʻi he genome ha afa ʻoku fakafuofua ki he 20,470. Meimei ko e 35% ʻo c. elegans afa ʻoku ʻi ai e homologs ʻa e tangata. Ko e meʻa fakaofo, he kuo toutou fakaha ʻa e afa ʻo e tangata ke fetongi ʻaki ʻenau C. elegans homologs ʻi he taimi naʻe fakafeʻiloaki ai ki he C. elegans. ʻI he tafaʻaki ʻe taha, ʻe lava ke ngaue tatau pe ʻa e tokolahi ʻo e c. elegans afa ki mammalian afa.

ʻOku approx ʻa e lifespan ʻo C. elegans. uike ʻe 3 pea ʻoku ʻi ai ha ngaahi tuʻunga moʻui ʻe ono: embryogenesis (tuʻunga ʻo e foʻimoa), ngaahi tuʻunga larval ʻe fa (L1 ki he L4), pea mo e tuʻunga ʻo e kakai lalahi. Ko e nematodes fofoa mei he fuaʻimoa ko L1 sivi kau ai ha ngaahi selo ʻe 560. ʻOku hoko ʻa e tupulaki ʻi he lolotonga ʻo e larval takitaha ʻi he vaʻa toʻotoʻo pea mo e hypertrophy toʻotoʻo. Cuticular molting punctuates he siteisi larval takitaha. Kapau ʻoku ʻi ai ha ngaahi tukunga fefeka ʻo e ʻatakai ʻoku fakaʻilongaʻi ki hono fakatupulaki ʻo e ʻuanga ʻoku ʻikai malava ke poupouʻi ʻe he ngaahi tukunga ʻa e kakai lalahi, C. elegans lava ʻo liliu ʻene fakalakalaka mo faʻu ha l3 ʻe taha larval siteisi, ʻa ia ʻoku ʻalu ai ʻa e sivi ki ha siteisi dauer. ʻI he tuʻunga ko ʻeni, ʻoku hoko ʻa e fanga monumanu ko ha loto-hohaʻa mo ha moʻui fuoloa pea ʻe lava ke nau moʻui ʻi ha mahina ʻe tolu ki he hiva. Dauer sivi fakamavaheʻi kinautolu mei he faingataʻa ʻaki hono silaʻi fakatouʻosi ʻenau buccal mo anal cavities, holomui mei heʻenau gut, pea fakamoʻui ha polokalama tukufakaholo daf-16/FOXO, ʻa ia ʻoku kau ai ha ngaahi meʻa kehe ke fakahaaʻi ʻaki ha kiutikolo pau dauer. (FF. Henderson et Al., 2006)

C. elegans Dauer sivi

Ko Dauer sivi ko e foʻi lea ia ki nematode sivi, ʻa ia naʻe hu ki ha tuʻunga fakalakalaka makehe. Ther ʻoku tautautefito hono fakaʻaongaʻi ʻo e foʻi lea ko e "Dauer sivi" ki he fanga ʻuanga ʻo e famili rhabditids kau ai ʻa Caenorhabditis elegans. Ko e foʻi lea ko e "Dauer" ko e tupuʻanga ia ʻo Siamane pea ʻoku ʻuhinga ia ki he "loloa” ʻi he ʻuhinga ʻo e “ha vahaʻa taimi ". Dauer sivi ʻalu ki ha faʻahinga stasis pea lava ke ne moʻui ʻi ha ngaahi tuʻunga fakamamahi. Kapau ʻoku hu mai ha larva ki he siteisi dauer ʻoku fakafalala ia ki he ngaahi tuʻunga ʻo e ʻatakai. ʻOku ako lahi ʻa Dauer sivi ʻi he paiolosii koeʻuhi he ʻoku fakahaaʻi ʻe he laravae ha malava makehe ke moʻui ʻi ha ngaahi ʻatakai fefeka pea moʻui ʻi ha vahaʻa taimi loloa. Hange ko ʻeni, ʻe lava ke moʻui ʻa C. elegans dauer sivi ʻo aʻu ki ha mahina ʻe fa, ʻo loloa ange ia ʻi he ʻavalisi ʻo e lifespan ʻi ha uike nai ʻe tolu lolotonga e fakalakalaka angamaheni.