Basa rekuongorora kubatana kwemapuroteni nemapuroteni

Alpha Lifetech inogonawo kupa vatengi bvunzo dzeprotein, ongororo yekudyidzana kweprotein, bvunzo yekudyidzana kweprotein-protein (CO-IP, Western Blot, Chromatin immunoprecipitation assay), uye bvunzo yekushanda kweprotein kuti isangane nezvinodiwa zvekuyedza zvevatengi vakasiyana.

TAURA NESU

Basa rekuongorora kubatana kwemapuroteni nemapuroteni

Alpha Lifetech yave ichinyanya kushanda mukuyedza mapuroteni kwemakore akawanda ichivaka puratifomu yakanaka yekuyedza mashandiro emapuroteni, uye iine hunyanzvi hwemhando dzakasiyana dzekuongorora mashandiro emapuroteni, izvo zvinogona kuchengetedza nguva yekutsvaga kwesainzi kana kutsvagisa mapurojekiti uku ichipa mhedzisiro yakagadzikana.

Nhanganyaya yeKuedza Kubatana kweProtein-Protein

Mapuroteni ndiwo mamorekuru akakosha zvikuru muhupenyu hwese achipindura mirairo yakachengetwa muzvinhu zvemajini. Kazhinji, mapuroteni anoita mabasa awo nekuita sevatambi vanobatanidzwa nemamwe mapuroteni, izvo zvinonyanya kuoneka kana basa repuroteni rikaongororwa mumamiriro chaiwo esero. Kuziva kudyidzana kwemapuroteni kwakakosha zvikuru pakuferefeta biochemistry yepuroteni yega yega. Kuongorora kudyidzana kwemapuroteni kwemhando dzakajairika seinotevera:

Kuedzwa kweCo-immunoprecipitation

Nheyo yenzira ye co-immunoprecipitation ndeyekusanganisa protein yakatarwa, antibody chaiyo, uye mapuroteni A/G magnetic beads kuti ifukidzwe, supernatant yeprotein isina kusungwa inoraswa kuburikidza nekudhonzwa kwema magnetic beads ari pa magnetic rack, uye ma magnetic beads anogezwa kubvisa protein nezvimwe tsvina kunze kwekunge zvakasungwa zvakananga.

Mufananidzo 1. Dhayagiramu yekuongorora Co-IP.(Mashoko ekutsvaga: Kuyedzwa kweCoimmunoprecipitation - PubMed (nih.gov))

Kuyedza Kudhonza-pasi

Kuyedza kunodonhedzwa pasi ndekwekugadzirisa puroteni inozivikanwa pabead remagineti uye kuwedzera chinhu chinofanira kuonekwa. Iyo eluent inoonekwa kuti mapuroteni anosangana.

Muna 1988, Smith nevamwe vake vakachenesa GST fusion protein, iyo yakashandiswa mukuyedza kudonhedza pasi uye inonzi Gst pull down iyo inoreva kuwedzera GST tag kune target protein, kubata protein inobatanidza kuburikidza neGSH, kujekesa kubatana, uye kuisimbisa kuburikidza neWB assays.

Mufananidzo 2. Dhiyagiramu yekuongorora GST pull-down.(Mashoko ekutsvaga: GST Pull-Down Assay yekudzidza PIF4 Binding In Vitro - PubMed (nih.gov) )

Chinangwa chake ndechekushandisa ma ions esimbi akadai se nickel kana cobalt senzira yekuchenesa mapuroteni kuburikidza ne affinity chromatography, uye kutsanangura kubatana kuburikidza nekuyedza kweWB kuti zviongororwe.

Pamusoro pezvo, kune DNA Pull-down (Protein DNA binding assay), RNA Pull-down (Protein rna interaction assay), uye small molecule pull-down assays.

Kuyedzwa kweDNA (DNA binding assay) inzira yekuona kubatana kweDNA neprotein muropa. Kana mapuroteni chaiwo achibatana neDNA chaiyo kwakaonekwa nekuyedzwa kweWB; Zvidimbu zveDNA zvisingazivikanwe zvinobatana nemapuroteni, izvo zvidimbu zveDNA zvinozivikanwa nekuona MS.

Kuyedzwa kweRNA Pull-down (Protein rna interaction assay) inzira yekuona kubatana kweRNA nemapuroteni muropa. Kana mapuroteni chaiwo achibatana neRNA yakatarwa kwakaonekwa nekuyedzwa kweWB; Zvidimbu zveRNA zvisingazivikanwe zvinobatana nemapuroteni, izvo zvidimbu zveRNA zvinozivikanwa nekuona MS.

Kuyedzwa kweSM pull-down (kuyedzwa kwemamorekuru madiki) kunogona kuwana mapuroteni akananga anobatana nemamorekuru madiki, uye kana akabatanidzwa neMS, mapuroteni madiki akanangana nemamorekuru anogona kuongororwa nemazvo, ayo anogona kukamurwa kuita nzira yekuisa mavara mu vitro uye nzira yebiological orthogonal.

Kuedza kweWB

Kuedza kweWB (kunozivikanwawo seWestern Blot experiment kana Western blot), chinhu chikuru ndechekuita kweantigen neantibody, musimboti wekutanga ndewekuparadzanisa mapuroteni akacheneswa kuburikidza neSDS polyacrylamide gel electrophoresis uye kuaendesa kune solid phase carrier (senge PVDF membrane, NC membrane) kuburikidza ne membrane transfer (yakanyorova kana kuti yakawoma zvishoma). Mushure mekuvharika (uchishandisa BSA, buttermilk, nezvimwewo), primary antibody inoshandiswa kubatanidza zvakananga neprotein, uye fluorescein-labeled secondary antibody inowedzerwa mushure mekusuka firimu. Nekusunga antibody yechipiri neprimary antibody, target protein inogona kuonekwa ne substrate color development uye chemiluminescence. Inowanzo shandiswa kuona kana protein chaiyo inoratidzwa mumuenzaniso uye kuongorora zvakajeka huwandu hwayo hwekutaura.

Kuenzanisa Nzira yeKubatana kweMapuroteni neMapuroteni

Kuyedzwa kweprotein pull-down kwakafanana neCo-IP assays, dzese dziri dzekuongorora mapuroteni anosangana. Musiyano uripo pakati penzira mbiri idzi ndewekuti Co-IP assays inowana mapuroteni anosangana emapuroteni anozivikanwa, ane chokwadi chakati asi haagone kusimbisa kana mapuroteni anosangana achibatana zvakananga kana zvisina kunanga. Kuyedzwa kwePull-down ndekwekutsvaga puroteni isingazivikanwe ichibatana nepuroteni inozivikanwa. Inogona kusimbisa zvakananga kana puroteni inozivikanwa ichibatana nepuroteni yakawanikwa asi haigone kuziva mamiriro ekubatana mumuviri.

Tekinoroji yeCo-immunoprecipitation inoshandiswa kuongorora kana paine kudyidzana pakati pemapuroteni maviri kana anopfuura, zvichibva pakuyedza kweimmunoprecipitation, Kana tichienzanisa nedzimwe ongororo yekudyidzana kwemapuroteni ehunyanzvi hwekuyedza (GST Pull-down, Western Blot, Chromatin immunoprecipitation assay, nezvimwewo), tekinoroji yeCo-IP ine hunyanzvi hwakanyanya, kunzwa, uye kudzokorora kwakanyanya, izvo zvinogona kudzivirira kupindira kwekubatanidza kusiri kwakananga uye kuratidza kudyidzana kwemapuroteni ari mumamiriro echisikigo.

	GST Dhonza pasi	Co-IP	WB
Zvakanakira	* Zviri nyore kushandisa *Yakakodzera kucheneswa kwemapuroteni akasiyana-siyana	Kuongorora kudyidzana kwemukati memuviri Inogona kuonekwa neprotein iri pasi pemvura	Kunyatsojeka kwakasimba Hunhu uye huwandu mu vitro
Muganho	Pamwe kubatana kusina kujeka Kuwedzera kusimbiswa kwekudyidzana kunodiwa	Kuvimba zvakanyanya nema antibodies Kusunga kusina kujeka kunogona kuitika	* Zvinotora nguva *Zvakaoma kushandisa pakuongorora mapuroteni akawanda
Muenzaniso weKushandisa	Ziva kudyidzana Mapuroteni akacheneswa	Ongorora kuchinjirwa kwechiratidzo Maitiro ezvirwere	Kuongororwa kunotevera kwekudyidzana kweprotein-protein, protein-DNA, uye protein-RNA