prou
Lihlahisoa
RNA (dsRNA) ea likhoele tse peli ELISA KIT HCP0033A Setšoantšo se Hlahang.
  • RNA (dsRNA) ea likhoele tse peli ELISA KIT HCP0033A

RNA (dsRNA) ea makhalo a mabeli ELISA KIT


Nomoro ea katse: HCP0033A

Pakete: 48T/96T

Setsi sena ke Enzyme-Linked Immunosorbent Assay (ELISA) e kopantsoeng le tsamaiso ea biotin-Streptavidin.

Tlhaloso ea Sehlahisoa

Lintlha tsa sehlahisoa

Setsi sena ke Enzyme-Linked Immunosorbent Assay (ELISA) e kopanyang le biotin-Streptavidin system, bakeng sa tekanyo ea bongata ba dsRNA ka bolelele bo ka holimo ho 60 base pairs(bp), ho sa tsotellehe tatellano.Letlapa le kentsoe pele ka anti-dsRNA antibody.dsRNA e teng sampoleng ea eketsoa 'me e tlama ho li-antibodies tse koahetsoeng ka liliba.Ebe biotinylated anti-dsRNA antibody ea eketsoa ebe e tlama ho dsRNA sampole.Ka mor'a ho hlatsoa, ​​​​HRP-Streptavidin e kenngoa 'me e tlama ho anti-dsRNA antibody ea Biotinylated.Ka mor'a ho qhoqhoa, HRP-Streptavidin e hlatsuoa.Ebe tharollo ea TMB substrate ea eketsoa le ho hlophisoa ke HRP ho hlahisa sehlahisoa sa 'mala o moputsoa se fetohileng mosehla kamora ho eketsa tharollo ea acidic stop.Boima ba 'mala o mosehla bo lekana le palo ea dsRNA e hapiloeng ka poleiti.Monyetla o lekanngoa ho 450 nm.


  • E fetileng:
  • E 'ngoe:

  • Kopo

    Sesebelisoa sena ke sa tekanyo ea bongata ba dsRNA e setseng.

      

    Likarolo tsa Kit

     

    Likaroloana

    HCP0033A-1

    HCP0033A-2

    1

    Elisa Microplate

    8x6

    8 × 12

    2

    Sethibela-mafu sa ho Fumana Biotinylated (100x)

    60μL

    120μL

    3

    Hrp-Streptavidin (100x)

    60μL

    120μL

    4

    Dilution Buffer

    15mL

    30 ml

    5

    Tharollo ea Tmb Substrate

    6mL

    12mL

    6

    Emisa Tharollo

    3mL

    6mL

    7

    Sekhahla sa ho Hlatsoa se Tebileng (20x)

    20mL

    40mL

    8

    E tloaelehileng (UTP, 5ng/μL)

    7.5μL

    15μL

    9

    E tloaelehileng (pUTP,5ng/μL)

    7.5μL

    15μL

    10

    E tloaelehileng (N1-Me-pUTP, 5ng/μL)

    7.5μL

    15μL

    11

    Tloaelehileng (5-OMe-UTP, 5ng/μL)

    7.5μL

    15μL

    12

    STE Buffer

    25mL

    50mL

    13

    Plate Sealer

    2 likotoana

    4 likotoana

    14

    Buka ea litaelo le COA

    1 khopi

    1 khopi

     

    Polokelo le Botsitso

    1. Bakeng sa khiti e sa sebelisoeng: Thepa eohle e ka bolokoa ho 2 ~ 8℃ bophelong ba shelofo.Leseli le matla le lokela ho qojoa bakeng sa botsitso ba polokelo.

     

     

    2. Bakeng sa lisebelisoa tse sebelisitsoeng: Hang ha microplate e buloa, ka kopo koahela liliba tse sa sebelisoeng ka plate sealer 'me u khutlele mokotleng oa foil o nang le pakete ea desiccant, zip-seal mokotla oa foil' me u khutlele ho 2 ~ 8℃ kapele kamoo ho ka khonehang ka mor'a hore u sebelise.Li-reagents tse ling li lokela ho khutlisetsoa ho 2 ~ 8 ℃ kapele kamoo ho ka khonehang kamora ho sebelisoa.

     

    Lintho Tse Hlokahalang Empa li sa Fanoe

    1. Sebali sa Microplate se nang le 450 ± 10nm filthara (ho molemo haeba e ka lemoha ho 450 le 650 nm wavelength).

    2. Microplate shaker.

    3. Litlhahiso tse se nang RNase le li-tubes tsa centrifuge.

     

    Operation Flowchart

     ""

     

     

    Pele O Qala

    1. Tlisa lisebelisoa tsohle tsa kit le lisampole mocheso oa kamore (18-25℃) pele o sebelisoa.Haeba khiti e ke ke ea sebelisoa ka nako e le 'ngoe, ka kopo nka likhoele le li-reagents bakeng sa liteko tsa hajoale, 'me u siee likhoele tse setseng le li-reagents maemong a hlokahalang.

    2. Hlatsoa buffer: hlapolla 40mL ea 20× concentrated bafa ea ho hlatsoa ka 760mL ea metsi a hloekisitsoeng kapa a hloekisitsoeng ho lokisa 800mL ea 1× bafa ea ho hlatsoa.

    3. Maemo: spin kapa centrifuge tharollo ea stock pele e sebelisoa.Khakanyo ea litekanyetso tse 'ne tse fanoeng ke 5ng/μL.Bakeng sa maemo a UTP le pUTP dsRNA, ka kopo hlapolla tharollo ea setoko ho 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL ka STE buffer ho hula mothinya o tloaelehileng.Bakeng sa litekanyetso tsa N1-Me-pUTP dsRNA, ka kopo hlapolla tharollo ea setoko ho 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL ka STE buffer ho hula mothinya o tloaelehileng.Bakeng sa maemo a 5-OMe-UTP dsRNA, ka kopo hlapolla tharollo ea setoko ho 4,2,1,0.5, 0.25,0.125,0.0625, 0pg/μL ka STE buffer ho hula mothinya o tloaelehileng.Re khothaletsa hore litekanyetso li hlakolloe joalo ka lichate tse latelang:

     

    Litekanyetso tsa N1-Me-pUTP dsRNA

     

    Che.

     

    Qeto ea ho qetela.

    (pg/μL)

    Taelo ya dilution

    STE

    buffer

     

    maemo

     

     

    A

     

    B

    C

    D

    E

    F

    G

    H

    100

     

    2

     

    1

    0.5

    0.25

    0. 125

    0.0625

    0.0312

    0

    49μL

     

    490μL

     

    250μL

    250μL

    250μL

    250μL

    250μL

    250μL

    250μL

    1μL 5ng/μL e tloaelehileng

    10μL 100pg/μL

    tharollo

    250μL tharollo A

    250μL tharollo B

    250μL tharollo C

    250μL tharollo D

    250μL tharollo E

    250μL tharollo F

    /

    Bakeng sa maemo a 5-OMe-UTP dsRNA

     

    Che.

     

    Qeto ea ho qetela.

    (pg/μL)

    Taelo ya dilution

    STE

    buffer

     

    maemo

     

     

     

    A

     

    B

    C

    D

    E

    F

    G

    H

     

    100

     

    4

     

    2

    1

    0.5

    0.25

    0. 125

    0.0625

    0

     

    49μL

     

    480μL

     

    250μL

    250μL

    250μL

    250μL

    250μL

    250μL

    250μL

    1μL 5ng/μL

    maemo

    20μL 100pg/μL

    tharollo

    250μL tharollo A

    250μL tharollo B

    250μL tharollo C

    250μL tharollo D

    250μL tharollo E

    250μL tharollo F

    /

    4. Li-antibody tsa ho lemoha ha biotinylated le tharollo ea ho sebetsa ea HRP-streptavidin: ka bokhutšoanyane spin kapa centrifuge tharollo ea thepa pele e sebelisoa.Li hlapolle ho mahloriso a sebetsang ka buffer ea dilution.

    5. TMB substate: labalabela tekanyo e hlokahalang ea tharollo ka malebela a hloekisitsoeng 'me u se ke ua lahlela motsoako o setseng ka har'a vial hape.TMB substate e na le kutloelo-bohloko ho khanya, e se ke ea pepesetsa karolo e nyenyane ea TMB mabone nako e telele.

     

    Ho sebelisa protocol

    1. Etsa qeto ea palo ea li-strip tse hlokahalang bakeng sa tlhahlobo.Kenya likhoele ka har'a liforeimi hore li sebelisoe.Lithapa tse setseng tse sa sebelisoang tekong ena li lokela ho kenngoa ka mokotleng ka desiccant.Koala mokotla ka thata bakeng sa polokelo ea sehatsetsing.

    2. Kenya 100μL e 'ngoe le e 'ngoe ea dilution ea maemo a tloaelehileng, a se nang letho le lisampole ka har'a liliba tse loketseng.Koahela ka sealer plate.Eketsa 1hr mocheso oa kamore ka ho sisinyeha ka 500rpm.Mehlala e lokela ho hlapolloa ka STE buffer ho tsepamisa mohopolo bakeng sa tlhahlobo e nepahetseng.

    3. Mohato oa ho hlatsoa: Aspirate tharollo 'me u hlatsoe ka 250μL bafa ea ho hlatsoa selibeng se seng le se seng ebe u e tlohela hore e eme bakeng sa 30s.Lahla sekheo sa ho hlatsoa ka ho feletseng ka ho phunya poleiti holim'a pampiri e monyang.Hlatsoa ka ho feletseng makhetlo a 4.

    4. Kenya 100μL ea tharollo e sebetsang ea antibody e lemohang biotinylated selibeng ka seng.Koahela ka plateer sealer.Eketsa 1hr mocheso oa kamore ka ho sisinyeha ka 500rpm.

    5. Pheta mohato oa ho hlatsoa.

    6. Kenya 100μL ea tharollo e sebetsang ea HRP-streptavidin selibeng ka seng.Koahela ka plateer sealer.Eketsa metsotso e 30 ka mocheso oa kamore ka ho sisinyeha ho 500rpm.

    7. Pheta mohato oa ho hlatsoa hape.

    8. Kenya 100μL ea tharollo ea TMB substrate selibeng ka seng.Koahela ka plateer sealer.Eketsa metsotso e 30 ho RT Sireletsa leseli.Mokelikeli o tla fetoha o moputsoa ka ho eketsoa ha tharollo ea substrate.

    9. Kenya 50μL ea tharollo ea ho emisa selibeng ka seng.Mokelikeli o tla fetoha mosehla ka ho eketsoa ha tharollo ea ho emisa.Ebe u tsamaisa sebali sa microplate 'me u tsamaise tekanyo ea 450nm hang-hang.

     

    Palo ea Liphetho

    1. Karolelano ea ho bala habeli bakeng sa boemo bo bong le bo bong, taolo, le lisampole ebe o tlosa palohare ea zero e tloaelehileng ea tekano ea mahlo.Theha mokoloko o tloaelehileng o monyehang holim'a axis e otlolohileng(Y) le dsRNA e tšekaletseng (X)axis).

    2. Ho kgothaletswa ho etsa lipalo ka software e thehiloeng ho curve-fitting software e kang curve expert 1.3 kapa ELISA Calc ka 5 kapa 4 parameter non-linear fit model.

    Tshebetso

    1. Boikutlo:

    moeli o tlase oa ho lemoha: ≤ 0.001pg/μL (bakeng sa UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (bakeng sa 5-OMe-UTP-dsRNA).

    moeli o tlase oa palo: 0.0156 pg/μL (bakeng sa UTP-, pUTP-dsRNA), 0.0312 pg/μL (bakeng sa N1-Me-pUTP-dsRNA), 0.0625 pg/μL (bakeng sa 5-OMe-UTP-dsRNA).

    2. Ho nepahala: CV ea Intra-Assay ≤10%, CV ea Inter-Assay ≤10%

    3. Pholiso:80%~120%

    4.Linearity:0.0156-0.5pg/μL(forUTP-,pUTP-dsRNA)0.0312-1pg/μL(forN1-Me-pUTP dsRNA),0.0625-1pg/μL(bakeng sa 5-OMe-UTP-dsRNA).

     

    Ho nahanela

    1. TMB karabelo mocheso le nako ke tsa bohlokoa, ka kopo li laole ho latela litaelo ka tieo.

    2. E le hore u finyelle tlhahiso e ntle ea assay reproducibility le kutloisiso, ho hlatsoa ka nepo lipoleiti ho tlosa li-reagents tse feteletseng tse sa sebetsaneng ke tsa bohlokoa.

    3. Li-reagents tsohle li lokela ho tsoakoa ka ho feletseng pele li sebelisoa le ho qoba li-bumbles nakong ea sampuli kapa li-reagents tse eketsehileng.

    4. Haeba likristale li entsoe ka har'a sekontiri sa ho hlatsoa (20x), se futhumala ho isa ho 37 ℃ 'me u kopanye ka bonolo ho fihlela likristale li qhibiliha ka ho feletseng.

    5. Qoba tlhahlobo ea lisampole tse nang le Sodium Azide (NaN3), kaha e ka senya tšebetso ea HRP e bakang khakanyo e tlase ea palo ea dsRNA.

    6. Qoba tšilafalo ea RNase nakong ea tlhahlobo.

    7. Tekanyetso/sample, antibody e lemohang le SA-HRP le eona e ka etsoa ho RT ntle le ho sisinyeha, empa sena se ka etsa hore kutloisiso ea ho lemoha e fokotsehe ka nako e le 'ngoe.Tabeng ena, re khothaletsa UTP le pUTP dsRNA maemo a lokela ho hlapolloa ho tloha 2pg/μL, N1-Me-pUTP dsRNA maemo a lokela ho hlapolloa ho tloha ho 4pg/μL le 5-OMe-UTP dsRNA standard e lokela ho hlapolloa ho tloha ho 8pg/μL.Ho phaella moo, kenya tharollo ea ho sebetsa ea HRP-streptavidin bakeng sa 60min mocheso oa kamore.Se ke oa sebelisa shaker ea flask, hobane flask shaker e ka baka sephetho se sa nepahalang.

     

    Sephetho se tloaelehileng

    1. Lintlha tse tloaelehileng tsa curve

    ho tsepamisa maikutlo

    (ts/μl)

    N1-Me-pUTP e fetotsoe maemo a dsRNA

    OD450-OD650(1)

    OD450-OD650(2)

    KAKARETSO

    2

    2.8412

    2.7362

    2.7887

    1

    1.8725

    1.9135

    1.8930

    0.5

    1.0863

    1.1207

    1.1035

     

     ""

    0.25

    0.623

    0.6055

    0.6143

    0.125

    0.3388

    0.3292

    0.3340

    0.0625

    0.1947

    0.1885

    0.1916

    0.0312

    0. 1192

    0.1247

    0.1220

    0

    0.0567

    0.0518

    0.0543

    2. Palo e tloaelehileng ea curve

    3. Lebelo la ho lemoha liner: 0.0312- 1pg / μL

    Ho tsepamisa maikutlo (pg/μl)

    OD450-OD650

    1

    1.8930

    0.5

    1.1035

    ""

    0.25

    0.6143

    0.125

    0.3340

    0.0625

    0.1916

    0.0312

    0.1220

    Ngola molaetsa wa hao mona mme o re romele wona