Qhov txawv ntawm Northern Southern thiab Western Blotting

2024 Tus sau: Alex Aldridge | [email protected]. Kawg hloov kho: 2023-12-17 13:40

Qhov Tseem Ceeb Qhov txawv - Northern vs Southern vs Western Blotting

Kev kuaj xyuas cov kab ke tshwj xeeb ntawm DNA, RNA, thiab cov proteins yog qhov tseem ceeb rau ntau hom kev tshawb fawb hauv Molecular biology. Gel electrophoresis yog cov txheej txheem uas cais DNA, RNA, thiab cov proteins raws li lawv qhov ntau thiab tsawg. Los ntawm cov gel profiles, tshwj xeeb DNA ib ntus, RNA ib ntus, lossis cov protein raug kuaj pom los ntawm cov txheej txheem tshwj xeeb hu ua blotting thiab hybridization nrog cov ntawv lo sojntsuam. Muaj peb hom kev blotting xws li yav qab teb, sab qaum teb thiab sab hnub poob. Qhov sib txawv tseem ceeb ntawm sab qaum teb sab qab teb thiab sab hnub poob blotting yog nrog hom molecule nws pom los ntawm cov qauv. Southern blotting yog ib txoj hauv kev uas kuaj pom cov DNA tshwj xeeb los ntawm cov qauv DNA. Northern blotting yog cov txheej txheem uas kuaj pom ib qho RNA ib ntus los ntawm tus qauv RNA. Western blotting yog ib txoj hauv kev uas kuaj pom cov protein tshwj xeeb los ntawm cov qauv protein.

Dab tsi yog Southern Blotting?

Southern blotting txheej txheem yog tsim los ntawm E. M. Southern hauv 1975 rau kev txheeb xyuas cov DNA tshwj xeeb los ntawm cov qauv DNA. Qhov no yog thawj txheej txheem blotting qhia hauv molecular biology. Nws tau ua kom pom cov noob tshwj xeeb los ntawm DNA, cov kab mob tshwj xeeb los ntawm DNA, thiab lwm yam. Muaj ob peb kauj ruam koom nrog hauv cov txheej txheem blotting yav qab teb. Lawv muaj raws li nram no.

DNA cais tawm ntawm cov qauv thiab zom nrog kev txwv endonucleases.
Digested qauv yog cais los ntawm Agarose gel electrophoresis.
DNA tawg nyob rau hauv cov gel yog denatured rau hauv ib leeg los ntawm kev siv cov tshuaj alkaline.
Ib leeg tso DNA raug xa mus rau nitrocellulose lim membrane los ntawm kev hloov pauv capillary.
Hloov DNA yog tsau rau ntawm daim nyias nyias mus tas li.
Tsau DNA ntawm daim nyias nyias yog hybridized nrog cov ntawv soj ntsuam.
Unbound DNA yog ntxuav tawm ntawm daim nyias nyias los ntawm kev ntxuav.
X-ray zaj duab xis raug rau daim nyias nyias thiab npaj autoradiograph.

Southern blotting yog siv rau ntau yam ntawm molecular biology. Nws muaj txiaj ntsig zoo hauv RFLP daim ntawv qhia, kev tshawb fawb forensic, DNA methylation hauv cov noob qhia, nrhiav pom cov noob hloov pauv hauv cov kab mob caj ces, DNA ntiv tes, thiab lwm yam.

Qhov txawv ntawm Northern Northern thiab Western Blotting - 1

Daim duab 01: Southern Blotting Technique

Dab tsi yog Northern Blotting?

Northern blotting yog ib txoj hauv kev tsim los tshawb xyuas qhov RNA ib ntus lossis mRNA ib ntus los ntawm cov qauv los kawm txog cov noob caj noob ces. Cov txheej txheem no tau tsim los ntawm Alwine, Kemp, thiab Stark hauv xyoo 1979. Nws txawv ntawm cov txheej txheem ua kua qaub thiab western blotting vim muaj ob peb kauj ruam. Txawm li cas los xij, cov txheej txheem no tseem ua los ntawm gel electrophoresis, blotting, thiab hybridization nrog cov ntawv sau tshwj xeeb thiab tshawb pom. Northern blotting txheej txheem yog ua raws li hauv qab no.

RNA yog muab rho tawm los ntawm cov qauv thiab sib cais los ntawm gel electrophoresis.
RNA raug xa tawm los ntawm cov gel mus rau hauv daim nyias nyias thiab tsau.
Cov daim nyias nyias tau kho nrog cov ntawv sau npe sojntsuam npaj los ntawm cDNA lossis RNA (qhov kev sojntsuam yog ntxiv rau ib ntu tshwj xeeb hauv cov qauv).
Kev sojntsuam yog incubated nrog daim nyias nyias los khi nrog ib ntus.
Kev sojntsuam Unbound raug ntxuav tawm.
Hybridized fragments raug kuaj pom los ntawm autoradiograph.

Northern blotting yog ib qho cuab yeej tseem ceeb hauv kev tshawb nrhiav thiab ntsuas cov hybridized mRNA, kawm RNA degradation, kev ntsuam xyuas ntawm RNA ib nrab-lub neej, nrhiav pom ntawm RNA splicing, kawm gene qhia, thiab lwm yam.

Qhov txawv tseem ceeb - Southern, Northern vs Western Blotting

Daim duab 02: Northern Blotting

Western Blotting yog dab tsi?

Western blotting yog ib txoj hauv kev tshawb xyuas cov protein tshwj xeeb los ntawm cov protein sib xyaw los ntawm kev siv cov tshuaj tiv thaiv kab mob. Yog li ntawd, western blot tseem hu ua immunoblot. Cov txheej txheem no tau qhia los ntawm Towbin et al hauv xyoo 1979 thiab tam sim no nws niaj hnub ua hauv cov chaw kuaj ntshav rau kev tsom xam cov protein. Cov kauj ruam yog raws li nram no.

Proteins tau muab rho tawm los ntawm tus qauv
Cov protein sib cais los ntawm lawv qhov ntau thiab tsawg siv polyacrylamide gel electrophoresis
Sib cais molecules raug xa mus rau hauv PVDF membrane lossis nitrocellulose membrane los ntawm electroporation
Cov daim nyias nyias yog thaiv rau qhov tsis tshwj xeeb khi nrog cov tshuaj tiv thaiv
Hloov pauv cov protein yog khi nrog thawj cov tshuaj tiv thaiv kab mob (enzyme sau npe tshuaj tiv thaiv kab mob).
Cov daim nyias nyias tau ntxuav kom tshem tawm cov tshuaj tiv thaiv tsis tshwj xeeb
Cov tshuaj tiv thaiv kab mob raug kuaj pom los ntawm kev ntxiv cov substrate thiab kuaj pom cov xim precipitate tsim

Western blotting yog qhov tseem ceeb hauv kev tshawb nrhiav cov tshuaj tiv thaiv kab mob HIV hauv tib neeg cov ntshav ntshav. Western blot kuj tseem siv tau los ua qhov kev kuaj pom tseeb rau tus kab mob siab B thiab kuaj qhov tseeb rau tus kab mob nyuj vwm.

Qhov sib txawv ntawm Southern, Northern thiab Western Blotting

Daim duab 03: Western Blotting

Dab tsi yog qhov txawv ntawm Northern Southern thiab Western Blotting?

Northern vs Southern vs Western Blotting
Hom ntawm Molecule Detected
Northern Blotting	Northern blotting pom ib qho RNA ib ntus los ntawm tus qauv RNA.
Southern Blotting	Southern blotting ntes tau ib qho DNA ib ntus los ntawm DNA qauv.
Western Blotting	Western blotting pom cov protein tshwj xeeb los ntawm cov qauv protein.
Type of Gel
Northern Blotting	Qhov no siv Agarose / formaldehyde gel.
Southern Blotting	Qhov no siv Agarose gel.
Western Blotting	Qhov no siv Polyacrylamide gel.
Txoj Kev Blotting
Northern Blotting	Nov yog kev hloov pauv ntawm cov hlab ntshav.
Southern Blotting	Nov yog kev hloov pauv ntawm cov hlab ntshav.
Western Blotting	Nov yog kev hloov hluav taws xob.
Kev Tshawb Fawb Siv
Northern Blotting	cDNA lossis RNA sojntsuam sau npe radioactive lossis nonradioactively.
Southern Blotting	DNA sojntsuam tau sau npe xov tooj cua lossis tsis muaj xov tooj cua.
Western Blotting	Primary antibodies yog siv los ua kev sojntsuam.
Detection System
Northern Blotting	Qhov no yog ua tiav siv lub autoradiograph, lossis kuaj pom lub teeb lossis xim hloov.
Southern Blotting	Qhov no yog ua tiav siv lub autoradiograph, nrhiav pom lub teeb lossis xim hloov.
Western Blotting	Qhov no yog ua tiav los ntawm kev tshawb pom lub teeb lossis xim hloov.

Summary – Northern vs Southern vs Western Blotting

Blotting yog cov txheej txheem tshwj xeeb tsim los rau kev txheeb xyuas cov DNA, RNA lossis cov protein los ntawm cov qauv. Muaj peb txoj kev blotting cais, uas yog sab qaum teb, sab qab teb thiab sab hnub poob, txhawm rau txheeb xyuas ib hom molecule. Northern blotting txheej txheem yog tsim los xyuas kom meej RNA ib ntus los ntawm kev sib xyaw ntawm RNA. Cov txheej txheem blotting yav qab teb ua rau kev tshawb pom ntawm DNA ib ntus los ntawm cov qauv DNA thiab cov txheej txheem blotting sab hnub poob yog tsim los txheeb xyuas cov protein tshwj xeeb los ntawm cov protein sib xyaw.