Streptavidin Sigma CAS 9013-20-1

1. Tau paub streptavidin
Streptavidin (SA), ib qho protein uas zais cia tau los ntawm Streptomyces avidinii, muaj qhov hnyav molecular ntawm kwv yees li 65-66 kDa thiab qhov taw qhia isoelectric ntawm ib ncig ntawm 6.0. Nws yog tetramer protein uas tsim los ntawm plaub homologous subunits, txhua tus muaj ib qho kev ywj pheej biotin khi qhov chaw - lub ntsiab lus hais tias ib qho molecule ntawm streptavidin tuaj yeem khi rau plaub lub molecules ntawm biotin. Lub dissociation tas li (Kd) ntawm ob yog tsawg li ntawm 10 ⁻¹⁵ M, uas yog ib qho ntawm cov muaj zog paub tsis muaj covalent lom lom neeg sib cuam tshuam thiab tuaj yeem suav tias yog qhov tseeb tshaj plaws "molecular locking mechanism" hauv xwm.

Piv nrog avidin muab tau los ntawm qe dawb, nws muaj qhov zoo sib xws:

Nws tsis muaj cov kev hloov kho glycosylation, yog tag nrho cov hluav taws xob nruab nrab, yog li muaj qhov tsis tshua muaj siab heev uas tsis yog- tshwj xeeb khi nyob rau hauv lub cev nqaij daim tawv, huv si tom qab sim, thiab cov teeb liab zoo heev - rau - suab nrov piv. Tsis tas li ntawd, nws zoo heev rau denaturation thiab protease degradation, nrog rau nws muaj peev xwm tiv taus huab cua pH, siab kub (70-80℃C), organic solvents, thiab cov pa denaturing agents (xws li SDS thiab urea), tau hloov avidin thiab dhau los ua qhov tsis sib haum xeeb "tus qauv kub" hauv biotin binding systems.

Nws yog qhov tseeb nrog cov yam ntxwv zoo uas nws ci ntsa iab hauv thaj chaw xws li kev tshawb fawb txog lub neej, kev kuaj mob hauv vitro, tshuaj ntsuam xyuas tshuaj, thiab biosensing.

2. Kev sim tshuaj tiv thaiv kab mob
Daim ntawv thov nyob rau hauv daim teb ntawm kev tiv thaiv kab mob yog nws feem ntau classic thiab dav siv daim ntawv thov. Txij li thaum nws txoj kev loj hlob nyob rau hauv lub lig 1970s, lub biotin streptavidin system (BAS) tau dhau los ua lub hauv paus ntawm kev tiv thaiv kab mob thiab kev soj ntsuam xyuas vim nws cov ultra-siab affinity thiab cascade amplification nyhuv. Raws li kev txheeb cais, kwv yees li 85% ntawm cov kws tshuaj ntsuam xyuas chemiluminescence siv BAS raws li kev tshawb pom, qhia txog lawv txoj haujlwm tseem ceeb.

2.1 Enzyme linked immunosorbent assay (ELISA)

Hauv ELISA, horseradish peroxidase (HRP) lossis alkaline phosphatase (AP) tuaj yeem ua ke thiab cov enzyme tuaj yeem raug coj mus rau lub hom phiaj los ntawm biotinylated antibodies. HRP catalyzes TMB, DAB, los yog ECL luminescent substrates los tsim cov tshuaj muaj zog lossis kho qhov muag teeb liab, thaum AP catalyzes BCIP / NBT, PNPP thiab lwm yam substrates los tsim cov xim ruaj khov lossis chemiluminescence signals. Lub hom phiaj biotinylated molecule tuaj yeem khi rau ntau daim ntawv sau npe streptavidin, ua tiav ntau - theem teeb liab amplification thiab ua kom pom cov hom phiaj tsawg tsawg tau. Cov thev naus laus zis no tau siv dav hauv cov qhaub cij thiab kev sib tw ELISA, suav nrog cov xwm txheej xws li protein ntau, kuaj tshuaj tiv thaiv kab mob, kuaj kab mob, thiab lwm yam.

2.2 Protein immunoblotting (Western Blot)
Nyob rau hauv Western Blot, khi nrog biotinylated cov tshuaj tiv thaiv kab mob thib ob tuaj yeem txhim kho cov teeb liab siv ntawm cov hom phiaj ntawm cov yeeb yaj kiab hloov chaw. Piv nrog rau cov kab mob thib ob cov tshuaj tiv thaiv kab mob, BAS systems tuaj yeem ua tiav cov teeb liab muaj zog dua vim lawv cov khoom sib txuas tetravalent. Tsis tas li ntawd, vim qhov tsis tshua muaj - tshwj xeeb khi ntawm streptavidin, kev cuam tshuam yav dhau los tau txo qis, ua rau lawv tshwj xeeb tshaj yog tsim nyog rau kev soj ntsuam ntau ntawm cov protein ntau tsawg.

2.3 Immunohistochemistry (IHC) thiab Immunocytochemistry (ICC)
HRP sau npe lossis AP sau npe streptavidin yog cov khoom siv hluav taws xob nyiam rau IHC thiab ICC. HRP system yog tsim nyog rau cov kev sim uas yuav tsum tau muaj siab nrhiav kom tau rhiab heev, thaum lub AP system yog tshwj xeeb tshaj yog haum rau cov tshuab uas muaj HRP inhibitors (xws li sodium azide) los yog kev sim scenarios uas yuav tsum tau ntev - lub sij hawm kev loj hlob xim thiab teeb liab stability. Los ntawm kev khi biotinylated sojntsuam lossis cov tshuaj tiv thaiv rau streptavidin conjugates, lub hom phiaj molecules tuaj yeem raug nyob hauv cov ntaub so ntswg lossis cov qauv ntawm tes.

2.4 Immunofluorescence (IF) thiab nyob rau hauv situ hybridization (ISH)

Fluorescein sau npe streptavidin (xws li FITC Streptavidin, PE Streptavidin, APC Streptavidin) yog cov cuab yeej pom kev thoob ntiaj teb rau kev siv tshuaj tiv thaiv kab mob thiab hauv qhov chaw hybridization. FITC marker muaj qhov excitation / emission wavelength ntawm kwv yees li 494/519 nm thiab nthuav tawm ci ntsuab fluorescence; Qhov excitation/emission wavelength ntawm PE marker yog hais txog 565/578 nm, uas yog "tus qauv kub" rau ntau -xim cytometry tsom xam; Tus cim APC npog cov xim liab nyob deb, ua kom yooj yim ntau- xim duab. Cov cim fluorescent no tuaj yeem raug khi rau biotinylated antibodies lossis nucleic acid sojntsuam kom ua tiav qhov kev txheeb xyuas hauv cheeb tsam thiab nrhiav kom muaj nuj nqis ntawm lub hom phiaj molecules.

2.5 Flow cytometry (FACS)

Hauv kev khiav cytometry, fluorescently sau npe streptavidin (tshwj xeeb tshaj yog PE daim ntawv lo) yog siv los xyuas cov cell nto antigens sau nrog biotinylated antibodies. Nws qhov ci ntsa iab zoo heev muab lub teeb liab siab heev - rau - suab nrov piv, uas tuaj yeem kuaj pom cov neeg hauv xov tooj tau zoo nrog cov kab mob antigen qis lossis cov cim tsis muaj zog. Nws yog ib qho tseem ceeb reagent nyob rau hauv ntau -xim flow cytometry tsom xam.

2.6 Enzyme txuas Immunospot Assay (ELISPOT)
AP sau npe streptavidin ua tau zoo heev hauv ELISPOT thiab tsim nyog rau cov khoom-phase tsom xam thiab cov ntaub so ntswg / cell staining systems. Nws tuaj yeem ruaj khov catalyze substrate ntau lawm ntawm cov cim chaw suav tau rau ib zaug -cell theem cytokine secretion detection.

3. Purification ntawm cov protein thiab nucleic acids
3.1 Affinity chromatography thiab protein purification
Streptomycin yog qhov tseem ceeb ntawm kev sib koom ua ke chromatography packing. Tom qab biotin sau npe cov protein kom huv, nws yog tov nrog streptavidin agarose gel (xws li Streptavidin Agarose / Sepharose), thiab cov protein uas tau sau npe yuav zoo khi rau streptavidin ntawm qhov muab tub lim. Los ntawm kev ntxuav kom tshem tawm cov impurities, thiab tom qab ntawd ntxuav lub hom phiaj protein nyob rau hauv qhov tsim nyog (xws li boiling SDS-PAGE buffer, 0.1% SDS kev kho mob, los yog 95% formamide+10 mM EDTA kev kho mob ntawm pH 8.2), siab -purity khoom yuav tau.

Noj cov kev lag luam tsim tawm ntawm tib neeg cov tshuaj insulin ua piv txwv, siab -zoo streptavidin magnetic hlaws tuaj yeem khi ntau dua 1200 pmol ntawm biotin dawb ib milligram, thiab cov protein purity purity tuaj yeem ncav cuag ntau dua 95%.

3.2 Immunoprecipitation (IP) thiab chromatin immunoprecipitation (ChIP)
Daim ntawv thov ntawm streptavidin magnetic hlaws dai hauv immunoprecipitation zoo heev txhim kho kev sim ua haujlwm. Cov kws tshawb fawb tuaj yeem kho cov tshuaj tiv thaiv biotinylated mus rau streptavidin magnetic hlaws thiab co incubate lawv nrog cell lysate.

Cov tshuaj tiv thaiv tshwj xeeb ntes cov hom phiaj protein thiab nws cov protein sib cuam tshuam, ua rau "magnetic hlaws antibody target protein" complex. Nws tuaj yeem raug cais tawm sai sai nyob rau hauv qhov kev txiav txim ntawm ib qho chaw sib nqus sab nraud, nrog kev ua haujlwm yooj yim thiab siab rhiab heev. Hauv kev sim ChiP, streptavidin magnetic hlaws kuj tau siv los ntes biotinylated DNA protein complexes thiab kawm txog kev cuam tshuam ntawm cov proteins thiab DNA.

3.3 Rub tawm kev sim
Hauv kev kawm txog cov protein-kev sib cuam tshuam cov protein, cov hlaws sib nqus tuaj yeem ntes cov protein uas muaj biotinylated thiab tom qab ntawd "ntses" rau cov proteins ua ntej uas cuam tshuam nrog lawv. Piv txwv li, thaum kawm cov kev taw qhia tseem ceeb hauv cov qog hlwb, cov tshuaj tiv thaiv tshwj xeeb uas paub txog P53 protein yog immobilized ntawm streptavidin sib nqus hlaws thiab co incubated nrog qog cell lysate kom muaj txiaj ntsig zoo P53 thiab nws cov proteins cuam tshuam. Piv nrog rau cov txheej txheem tiv thaiv kab mob ib txwm muaj, cov hlaws sib nqus IP muaj qhov rhiab heev thiab qhov tshwj xeeb, thiab tuaj yeem ua kom muaj txiaj ntsig zoo rau cov protein ntau tsawg.

3.4 RNA rub- nqis
Cov hlaws sib nqus kuj tseem tsim nyog rau RNA rub- kev sim qis. Tom qab khi nrog streptavidin sib nqus hlaws, biotinylated RNA sojntsuam tuaj yeem ntes cov proteins uas cuam tshuam nrog RNA los ntawm cell lysates rau kev kawm RNA protein kev sib koom tes.
3.5 Ib leeg stranded DNA npaj thiab nucleic acid extraction
Nws tuaj yeem khi nrog biotinylated oligonucleotides rau kev npaj ntawm ib leeg stranded DNA thiab muaj txiaj ntsig extraction ntawm nucleic acids, ua lub luag haujlwm tseem ceeb hauv molecular cloning thiab genetic engineering.

4. High throughput detection thiab tshuaj ntsuam xyuas tshuaj
4.1. TR-FRET xeem platform
Nws tuaj yeem ua ke nrog cov luminescent chelates xws li europium chelates los ua cov teeb liab txuas thiab kev ntes cov molecular hauv TR-FRET (lub sij hawm- daws teeb meem fluorescence resonance zog hloov) kev kuaj xyuas qhov system. Piv txwv li, THUNDER ™ Lub europium sau npe streptavidin tshwj xeeb tsim los rau TR-FRET kev sim platform tuaj yeem ruaj khov rau ntau yam biotinylated hloov kho cov molecules thiab tau siv dav hauv kev tsom xam cov protein sib cuam tshuam thiab kev tshuaj xyuas ua ntej ntawm cov tshuaj hom phiaj.

Cov txheej txheem no yog homogeneous thiab siab -throughput, ua rau nws yog ib qho cuab yeej muaj zog rau kev txhim kho tshuaj niaj hnub.

4.2 Lub hom phiaj xa tshuaj
Nws cov biotin binding zog ua rau nws yog ib qho zoo tagnrho cov neeg nqa khoom rau cov tshuaj tsom xa cov tshuab. Los ntawm kev sib txuas cov tshuaj molecules nrog biotin thiab siv lub hom phiaj khi lub peev xwm ntawm streptavidin, kev xa tshuaj meej tuaj yeem ua tiav, txhim kho kev kho mob thiab txo cov kev mob tshwm sim.

4.3 Biosensors thiab Biochips

Nws yog cov khoom siv tseem ceeb rau kev tsim cov biosensors thiab biochips. Los ntawm immobilizing streptavidin nyob rau saum npoo ntawm cov nti raws li lub ntsiab lus ntes, biotin sau cov protein sojntsuam lossis cov tshuaj tiv thaiv tuaj yeem raug immobilized rau nws, ua tiav siab - dhau los thiab siab - kev kuaj pom. Piv txwv li, nyob rau hauv kev tshuaj ntsuam xyuas cov protein chips thaum ntxov rau cov kab mob plawv, streptavidin magnetic hlaws yog tsau rau ntawm nti substrate thiab siv los khi biotinylated protein probes. Qhov no tuaj yeem kuaj pom qhov txawv txav hauv cov neeg mob thaum ntxov ntawm tus kab mob thiab thaum cov ntshav cim qhia tau qis li cTnI ntawm 0.01 ng / mL, nce qhov kev kuaj mob los ntawm 2-3 teev piv rau cov kev kuaj mob ib txwm muaj.

5. Cell Biology thiab Imaging
5.1 Cell nto labeling thiab fluorescent cell sorting
Fluorescent sau npe streptavidin (xws li Vari Fluor 488 Streptavidin) yog siv dav rau kev sau npe ntawm tes thiab fluorescent cell sorting. Nws cov emission spectrum npog tag nrho cov pom thiab nyob ze -infrared lub teeb spectrum, thiab nyob rau hauv ntau- xim labeling thwmsim, nws yuav yooj yim nkag mus rau hauv lub nucleus thiab cytoplasmic qauv, daim ntawv lo lub hom phiaj proteins rau imaging thiab tsom xam.

5.2 Nanotechnology thiab Material Self Assembly
Nws tetravalent binding zog ua rau nws zoo tagnrho "choj" rau tus kheej - sib dhos ntawm nanomaterials. Los ntawm lub hom phiaj khi ntawm biotin streptavidin, ntau yam nanostructures tuaj yeem tsim los rau kev tshawb fawb txog kev sib raug zoo xws li kev kuaj kab mob lom neeg thiab kev saib xyuas ib puag ncig.

6. Anti streptavidin antibody
Nrog rau kev siv dav dav ntawm cov kab mob streptavidin biotin, ib qho teeb meem feem ntau tsis pom zoo tau tshwm sim - endogenous anti streptavidin antibodies (ASA) hauv cov qauv tuaj yeem cuam tshuam nrog kev kuaj pom. Kev tshawb fawb tau pom tias kev khi ntawm ASA rau SA hauv cov tshuaj tiv thaiv tuaj yeem cuam tshuam rau kev tsim kho ntawm cov kab mob antigen tshwj xeeb, uas ua rau muaj kev cuam tshuam qhov tseeb ntawm kev sib txuas, kev tshawb nrhiav, lossis kev kuaj mob.

Txhawm rau hais txog qhov kev sib tw no, cov khoom siv tshuaj tiv thaiv streptavidin antibody nrhiav tau tshwm sim, uas tuaj yeem siv los txheeb xyuas endogenous ASA hauv cov neeg mob kuaj thiab zam nws cuam tshuam nrog BAS system. Roche Custom Biotech tau tsim ib qho tsis muaj zog streptavidin mutant (Streptavidin rec. inactive, poly), uas tuaj yeem muab ntxiv rau cov txheej txheem tam sim no yam tsis muaj cov kauj ruam ntxiv los thaiv tshwj xeeb thiab tshem tawm ASA cuam tshuam, txhim kho qhov raug thiab tshwj xeeb ntawm cov txheej txheem tshawb nrhiav.

7. Biotin cuam tshuam
Txawm hais tias streptavidin biotin system muaj zog, nws ntsib qhov kev sib tw hnyav ntxiv - biotin cuam tshuam.

Thaum muaj qhov siab siab ntawm cov biotin dawb hauv cov qauv kuaj, nws sib tw nrog biotinylated antibodies rau qhov chaw khi ntawm streptavidin, uas ua rau muaj kev cuam tshuam kev tshawb pom.

Kev tswj hwm qhov ncauj ntawm 100 mg biotin tuaj yeem ua rau cov ntshav ntshav siab tshaj plaws ntawm 375-450 ng / mL; Cov neeg mob uas muaj ntau yam sclerosis tuaj yeem noj 300 mg / d ntawm qhov ncauj, thiab lawv cov ntshav plasma biotin tuaj yeem tshaj 1000 ng / mL; Kev noj tshuaj txhua hnub ntawm 500 mg ntawm biotin tuaj yeem ua rau muaj qhov ntau ntawm biotin hauv cov ntshav. Hauv Sandwich immunoassays, ntau biotin tuaj yeem tsim cov txiaj ntsig tsis zoo; Hauv kev sib tw immunoassays, nws tuaj yeem ua rau cov txiaj ntsig tsis zoo.

Ntau cov kev tshawb fawb tau pom tias, TSH, Thyroxine PTH, Endocrine kuaj xws li adrenocorticotropic hormone, prolactin, testosterone, cortisol, nrog rau cov cim qog thiab cov cim ua haujlwm ntawm lub plawv, txhua tus cuam tshuam rau qib sib txawv los ntawm biotin cuam tshuam. FDA tau tshaj tawm cov ntaub ntawv ceeb toom txuas ntxiv thiab cov lus qhia sib xws. Cov tswv yim daws teeb meem suav nrog: serial dilution ntawm cov qauv, hloov mus rau txoj kev tsis yog BAS xws li kua chromatography-mass spectrometry, rov kuaj dua tom qab biotin clearance (tsawg li 2 teev, ntev li 15 hnub lossis txawm lub hli), lossis siv cov khoom siv biotin dawb.