I-Hielscher Ultrasound Technology

I-Ultrasonic Lysis ye-Western Blotting

  • Ukususa entshonalanga kuyinkqubo yokuhlaziya yokutholakala kwamaprotheni akhethekile kwisampula yama-homogenate noma isiliva.
  • Ukuze usebenzise i-Western blot noma ukukala umsebenzi we-enzyme, izivivinyo eziningi zidinga ukuthola izinto (isib. Amaprotheni, i-DNA, izingcezu ze-subcellular) eziboshwe esitokisini.
  • I-Sonication iyindlela yokwethenjelwa futhi elula yokulawula ukuphazanyiswa kweseli ne-lysis.

I-Ultrasonic Cell Disruption

Izakhi zamaprotheni ezivela emathisini namaseli akhiqizwa yisinyathelo sokuqala samasu amaningi wezinto eziphilayo, eziphilayo kanye nokuhlaziya (IKHASI, ukuchithwa kwe-Western, i-ELISA, i-spectrometry mass, njll) noma amaprotheni ukuhlanzwa. Ukuze uthole amaprotheni aphezulu, isivinini sezinhlamvu kanye nezicubu kufanele sithinteke kahle / lysed kahle. Kungakhathaliseki ukuthi isitshalo sezitshalo noma izicubu zesilwane, indodanaication iyindlela yokulungisa i-lysate yakho kalula futhi esheshayo.

Izinzuzo ze-Sonication

  • Okusheshayo & ephumelelayo
  • umsebenzi olula
  • amaprotheni aphezulu
  • ukuphindaphinda / ukuphindaphinda
  • elawulwa kahle
  • i-scalable

I-Prounoprecipitation Protocol Ye-Western Immunoblotting

A. Ama-reagents

Ukuze ulungiselele izixazululo, sebenzisa amanzi ahlanzekile njengeMilli-Q.

  • I-1X i-Phosphate Buffered Saline (i-PBS)
  • I-1X ye-Cell Lysis Buffer: i-Tris engu-20mM (i-pH 7.5), i-NaCl engu-150mM, i-1 mM EDTA, i-1 mM EGTA, i-1% iTriton X-100, i-2.5mM i-sodium pyrophosphate, i-1 mM β-glycerophosphate, i-1 mM Na3VO4, i-1 μg / ml Leupeptin
    Okubalulekile: Engeza 1mM PMSF ngokushesha ngaphambi kokusetshenziswa.
  • 15 μl Amaphrotheni A + 15 μl Amaphrotheni G anele i-IP, kodwa ingaxhomeka ku-antibody yakho oyinhloko nesvolumu yesampula. Ungasebenzisa futhi amaprotheni ahlanganisiwe ngaphambilini A / G (isib. Amaprotheni A for rabbit IgG wehlisa futhi amaprotheni G for igg mouse IgG phansi)
  • I-3X SDS Sample Buffer: i-Tris-HCl engu-187,5mM (i-pH 6.8 ku-25 ° C), i-6% w / v i-SDS, i-30% ye-glycerol, i-150 mM i-DTT, i-0.03% i / i-bromophenol eluhlaza okwesibhakabhaka
Hielscher's VialTweeter is is´deal for the lysis of multiple samples

I-VialTweeter for ultrasonic isampula prep

Xhumana nathi! / Cela Us!





Sicela uqaphele wethu Inqubomgomo yobumfihlo.


Sonication yisinyathelo esibalulekile ngesikhathi sokulungiselela isampula

UP200St nge-tip-micro ye-sonication yesampula

B. Ukulungiswa kwamaLysates Cell

  • Vuna amangqamuzana. Ukuvuna amaseli ngaphansi kwezimo zokunakekela, susa imidiya bese ugeza amangqamuzana kanye ne-PBS ebandayo.
  • Susa i-PBS bese wengeza u-0.5 ml we-1X cell lysis buffer ku-plate ngayinye (u-10 cm) bese ubandakanya amapulethi eqhweni imizuzu emihlanu.
  • Khipha amaseli emaceleni bese udlulisela emathinini amancane we-microcentrifuge. Hlala phezu kweqhwa.
  • Bamba kabili imizuzwana engu-10 e-cold-immunoprecipitation buffer (i-IP buffer): i-TM-50 i-Tris-HCl [pH 7.4], i-NaCl engu-150mM, i-5 mM EDTA, i-0.1% NP-40 kanye ne-protease inhibitor mix). Ngokuba sonication, the I-VialTweeter noma ultrasonicator probe efana UP100H noma UP200Ht zifaneleke kakhulu.
  • I-centrifuge i-lysates ku-15,000 g imizuzu engu-10 ku-4 ° C.
  • Dlulisa i-supernatant ku-tube entsha. (Uma kunesidingo, i-lysate ingagcinwa ku- -80 ° C.)
  • Engeza i-antibody oyinhloko ku-supernatant. I-supernatant ne-antibody eyinhloko ihlanganiswa ngehora elingu-1 ° C ngaphansi kwe-4 ° C ngaphansi kokuphazamiseka okukhulu. I-antibody eyinhloko ivame ukungezwa ngemali engu-10x ngaphezulu egxilile kunasetshenziselwa ukubhujiswa entshonalanga. (Ungaqala ngo-1μg nge-100μL.)
  • I-supernatant isetshenziselwa ukuqhutshwa phambili ngenhlanganisela yemali elinganayo yeProtheni A-agarose (Invitrogen) neProtein G igarose enye ihora elilodwa.
  • Geza izikhathi ezintathu ze-agarose nge-IP buffer. Ngemuva kwalokho, ukhiphe amaprotheni anesibopho nge-SDS-PAGE ukulayisha i-buffer ngokushisa ngo-95 ° C imizuzu emihlanu.

C. Immunoprecipitation

  • Thatha i-200 μl cell lysate bese ufaka i-antibody eyinhloko. Hlanganisa ngobumnene ngobusuku obungu-4 ° C.
  • Engeza ubuhlalu be-protein A noma G obukhulu (20 μl we-50% bead slurry). Hlanganisa ngobumnene ngamahora angu-1-3 ku-4 ° C.
  • I-microcentrifuge imizuzwana engu-30 ngo-4 ° C. Geza i-pellet izikhathi ezinhlanu nge-500 μl ye-1X cell lysis buffer. Hlala phezu kweqhwa ngesikhathi sezindwangu.
  • Hlela kabusha i-pellet nge-20 μl ye-3X SDS yesampuli yesampula. I-Vortex, bese i-microcentrifuge imizuzwana engu-30.
  • Sushisa isampula ku-95-100 ° C ngamaminithi angu-2-5 no-microcentrifuge ngomzuzu owodwa ku-14,000 X g.
  • Layisha isampula (15-30 μl) kwi-gel ye-SDS-PAGE (12-15%).
  • Hlaziya isampula nge-Western blotting.

Xhumana Nathi / Cela Ukwaziswa Olwengeziwe

Khuluma nathi mayelana nezidingo zakho zokucubungula. Sizosikisela ukusetha nokulungiswa kwemingcele efanele yephrojekthi yakho.





Sicela uqaphele wethu Inqubomgomo yobumfihlo.


Izinqubo Zokunikezela Ezengeziwe

Western Blot ukuhlaziywa nge ultrasonicator UP50H

Ukulandelwa kweprotocol kusetshenziselwa ukutadisha uKriebisch et al. (2011):
Inani lamaprotheni lalihlukaniswe namaseli e-MC3T3-E1 aphathwa nge-1,25 (OH)2D3 (10-8 M) noma imoto. Amaseli ahlolwe nge-buffer ene-Tris HCl engu-50mM, i-pH 8 (Sigma-Aldrich); I-NaCl engu-150mM (i-Fisher Scientific); 0.1% i-sodium dodecyl sulfate (i-SDS) (i-Fisher Scientific); 1% IGEPAL CA-630 (Sigma-Aldrich) no-0.5% deoxycholate (Merck) ye-sodium. I-lysate yeseli yayihanjiswe nge-2 × 10 s kumjikelezo 1 no-amplitude 80 nge- UP50H i-Ultrasonic Processor (Hielscher, Ultrasound Technology, Teltow, eJalimane). Ngemuva kwaloko lo mkhakha wawuyi-centrifuged for 10 min ku-14,000 rpm kanti i-supernatant yayisetshenziselwa ukucima kwe-Western. Amakhilogremu angamashumi amabili nantathu ama-protein ayilisiwe kwisampuli ye-sampuli kanye ne-ejenti yokunciphisa (i-Invitrogen) bese ehlukaniswa yi-SDS-PAGE ngokusebenzisa ama-4-12% we-polyacrylamide gels (i-Invitrogen) futhi adluliselwe kumlenze we-nitrocellulose (ukunakekelwa kwempilo ye-GE). I-membrane ivinjelwe i-1 h nge-TBS (i-Tris-HCl engu-10 mM; pH 7.6; i-NaCl engu-150mM) equkethe i-1% ye-casein (Sigma-Aldrich) ne-1% iTris (1 M). Ngemuva kokuvimbela, i-membrane yayisetshenziswe ngobusuku obukhulu ngo-4 ° C ne-antibody eyinhloko (i-rabbit anti-human CBS 1/500, eyakhiwe ebhodini likaProfesa R. Banerjee, Ann Arbor, MI, eU.SA). Ukufakelwa kwe-peroxidase (HPR) e-horseradish (i-HPR) ekhonjisiwe (i-Dako) eyenziwe nge-1 h ekamelweni lokushisa. Zonke izibhamu zakhiwe yi-chemiluminescence ethuthukisiwe (i-Perkin Elmer).

Izincwadi / Izinkomba



Mayelana ne-Western Blotting

Amabhulogi yizinqubo zokuhlaziya lapho i-DNA, i-RNA kanye namaprotheni adluliselwa kubathwali ukuze bakwazi ukuhlukaniswa.
I-Southern blot isetshenziselwa ukuthola i-DNA, i-Northern Blot ye-RNA kanye ne-Western blot yama-protein.
Ukususwa kwe-Western kuthiwa yi-protein immunoblotting ngoba i-antibody isetshenziselwa ukuthola ngokuqondile i-antigen yayo. I-Western Blotting ingenye yezindlela ezibaluleke kakhulu zokuhlaziya ukuthola amaprotheni akhethekile kusampula. E-Western blot, amaprotheni akwazi ukuvimbela imfucumfucu ukuze abone ukuthi usebenzisa ama-antibodies monoclonal noma polyclonal.
Nge-SDS-polyacrylamide gel electrophoresis (i-SDS-IKHASI) amaprotheni omdabu ahlukaniswa isakhiwo se-3-D noma amaprotheni angama-denatured ngobude be-polypeptide. Ama-protein asetshenziselwa ulwelwesi (ngokuvamile i-nitrocellulose noma i-PVDF), lapho ahlotshiswe khona ngama-antibodies aqondene neprotheyini ehlosiwe. Isinyathelo se-gel electrophoresis sifakwe ekuhlaziyweni kwe-western blot ukuxazulula inkinga ye-cross-reactivity ye-antibodies.
Ngemuva kwalokho, amaprotheni ahlukanisiwe asulwa kumatrix (ikakhulukazi nge-membrane ye-nitrocellulose noma i-PVDF), lapho anesifo samagciwane. Ama-antibodies asebenza njengephrojekithi futhi akhethiwe ngqo kumaprotheni okuhlosiwe. Ukuhlaziywa kwendawo nokuqina komsakazo othize kubonisa imininingwane yezinhlelo zamaphrotheni okulindeleke kwisampula esinikeziwe. Ukususwa kwe-Western kungabona amaprotheni ahlosiwe angaphansi kwe-1ng ngenxa yokuxazulula okuphezulu kwe-gel electrophoresis nokucacile okuqinile nokuzwela okukhulu kwe-immunoassay. Indlela ye-Western blot isetshenziswa ku-biology yamangqamuzana, i-biochemistry, i-immunogenetics namanye amasimu okucwaninga kwamangqamuzana.
Amanye amasu ahlobene nawo ahlanganisa ukuhlaziywa kwe-dot blot, i-immunohistochemistry kanye ne-immunocytochemistry lapho amasosha omzimba asetshenziselwa ukuthola amaprotheni amathisini namaseli nge-immunostaining, ne-enzyme-ehlobene ne-immunosorbent testay (ELISA).