Thaum ib qho kev sim ua tsis tiav, koj puas tau taw tes ntiv tes ntawm cov twj siv hluav taws xob kim lossis cov twj paj nruag nyuaj? Tab sis qee zaum, qhov tseeb "ua txhaum" yuav nyob ntsiag to ntawm lub ces kaum - zoo li lub raj mis zoo li tsis muaj tseeb. Txawm hais tias nws yog inconspicuous, nws yog txoj sia rau kev tswj hwm qhov tseeb ntawm kev sim. Niaj hnub no, peb yuav uncover cov lus zais ntawm cov neeg saib xyuas no "tsis pom zoo".
Ⅰ. Buffer: Nws yog dab tsi? Vim li cas nws tsis tseem ceeb hauv chav kuaj?
Tsuas yog muab tso, ib qho tsis yog cov tshuaj tshwj xeeb uas tuaj yeem tiv thaiv qhov cuam tshuam ntawm cov khoom me me me me ntawm cov tshuaj muaj txiaj ntsig. Nws yog feem ntau muaj ntawm ib khub ntawm "cov neeg sib koom tes" - qhov tsis muaj zog acid / sodium acetate phosphate (nahh {acid ac ac), thiab lwm yam, thiab lwm yam, uas yog ib qho tshwm sim nyob rau hauv chaw soj nstuam.
Qaus peev: ruaj khov ph thiab tiv thaiv kev hloov!
Thaum cov khoom siv acidic me me lossis cov tshuaj Alkaline tau raug coj los ua qhov kev sim, muaj kev sib tw ua haujlwm, yog qhov ua rau muaj kev sib raug zoo hauv PHIC. Qhov peev xwm no yog qhov tseem ceeb rau ntau cov tshuaj tiv thaiv kab mob uas cia siab rau ib puag ncig hauv pH.
Ⅱ. Tact QIB: Ib qho tsis tseem ceeb ntawm cov protagonist thiab txhawb txoj haujlwm
1. "Lub Neej Kev Txhawb Txoj Haujlwm" ntawm kev sim biochemical:
Enzyme kev tshawb fawb:Feem ntau cov enzymes muaj cov kev ua tau zoo tshaj plaws tsuas yog nyob rau hauv cov phap nqaim. Buffers (xws li tris - hcl, PBS) muab "kev ua haujlwm ruaj khov" rau enzyme cov tshuaj tiv thaiv kom ntseeg tau.
Cov tshuaj muaj protein thiab tsom xam:Los ntawm tes lysis, ion txauv, aftinity chromerogrization thiab kev sib tw ntawm cov protein thiab kev tiv thaiv denaturation lossis nag lossis daus.
Nucleic acid manipulation:PCR cov kev tawm tsam, DNA / RNA rho tawm, enzyme zom, ligation, hyigation, hyigridization thiab lwm cov txheej txheem muaj kev nkag siab heev rau ph. Feem ntau siv buffers xws li te (tris - etta) tuaj yeem tiv thaiv lawv los ntawm nuclase degradation.
2. "Lullaby" ntawm Cell Catine:Nws tus kheej yog cov txheej txheem tsis sib xws (feem ntau cia siab rau sodium bicarbonate / carbon dioxide system). Ntxiv cov buffers ntxiv (xws li hepes) tuaj yeem muab cov hlwb nrog cov kua dej khov kho, saib xyuas cov pa roj carbon dioxide ntxiv.
3. Cov "stabilizing quab yuam" ntawm kev ntsuas anchistry:
Chromatographic sib cais:Hauv kev ua tau zoo ua kua chromatography (HPLC) thiab ion tswj cov nyhuv rau kev sib cais thiab acetates) xyuas kom muaj kev sib cais ntawm cov txheej txheem sib cais.
Cov ntsuas ntsuas hnub no:Qee qhov kev hloov pauv tseem ceeb yuav tsum tau nqa tawm ntawm lub pH tas mus li lossis qhia tau qhov kawg thaum lub pH tshwj xeeb tau mus rau lub luag haujlwm ruaj khov.
Npaj cov kev daws teeb meem:Tus nqi PHF ntawm ntau cov kev daws teeb meem cov qauv yuav tsum tau ua kom paub tseeb, thiab cov tsis tau ua yog lub hauv paus.
Ⅲ. Xaiv thiab siv: kev paub ntau
Xaiv txoj cai tsis xis nyob:
PKA tus nqi yog tus yuam sij:Cov kev ua tau zoo ntawm cov tsis muaj feem ntau yog nyob hauv qhov ntau ntawm nws pka ± 1 tus nqi. Nco ntsoov xaiv tus tsis nrog PKA tus nqi ze rau PHICT PHIC koj xav tau! Piv txwv li, phosphate (Pka ~ 7.2) feem ntau siv nyob rau hauv qhov chaw nruab nrab hauv nruab nrab, thiab tris (PMA ~ 8.1) feem ntau siv hauv Alkaline.
Kev sib haum:Txiav txim siab seb puas yog cov tsis sib haum tsis sib haum nrog koj cov tshuaj uas muaj kev sim siab (piv txwv li cov tshuaj tiv thaiv kab mob phosphatehlation, thiab borate yuav hnov mob nrog cov suab thaj).
Lub zog muaj zog thiab kub:Lub zog muaj zog cuam tshuam cuam tshuam enzyme kev ua si, solubility, thiab lwm yam .; Kev hloov qhov kub hloov tuaj yeem cuam tshuam ntau dua rau PKA tus nqi ntawm buffers xws li tris (tris muaj ntau pka kub coefficient ntawm kwv yees li -0.031 / degree).
Ⅳ. Cov npe ntawm cov tsis sib xws
1. PBS Tsis
PBS Tsis yog 1 × (ph7.4, PBS Tsis yog 10 × (PBS phosphate hmoov (xws li ua ntej hloov cov kab lis kev cai hauv nruab nrab); Diluting cov tshuaj tiv thaiv thiab ntxuav Elisa Daim Hlau hauv kev sim tshuaj tiv thaiv kab mob; Tswj cov osmotic thiab pH ruaj khov nyob hauv cov protein / nucleic acid sim, thiab lwm yam.
Dpbs tsis (tsis muaj calcium thiab magnesium), ntxuav cytometry hmoov (kom tsis txhob muaj calcium thiab magnesium cuam tshuam nrog enzyme kev ua); Diluting enzymes lossis ua ion -} Cov sim rhiab hauv cov roj ntsha (xws li qee qhov pccr kev), thiab lwm yam.
PBST tsis (1 ×), PBT tsis (10 ×): siv rau kev thaiv thiab ntxuav cov kauj ruam hauv Western blue lossis immunohistochemistry, txo tus nqi siv los, thiab lwm yam.
2. TBS Buffer Series
TBS Tsis (1 ×), TBS Tsis (10 ×): Kev sim ua kom tsis txhob muaj kev cuam tshuam ntawm kev txhaj tshuaj tiv thaiv kab mob (kom tsis txhob muaj qee yam tshuaj tiv thaiv kab mob); Txawb ob puag ncig pH hauv ib puag ncig hauv cov protein sim, thiab lwm yam.
3. HBSS Buffer Series
Cov tsis muaj (calcium thiab cov magnesium dawb, tshuaj ntsuab penol (calcium thiab magnesium dawb): xws li cov cytometry (xws li ntws cytometry); Luv -} Lub Cell Kev Txij Nkawm (xws li cov khoom siv xov tooj ntawm tes (calcium thiab magnesium koom nrog teeb liab transduction); Kev tsiv teb tsaws siab / ntxeem tau cov kev sim.
4. Tou buffer series
TE Buffer (1 ×), TE Buffer (10 ×): Dissolve thiab khaws tseg DNA / RNA chelection hlau ions rau inhibit nuclease kev ua ub no); Dilute DNA rau PCR, extraCaling thiab lwm yam kev sim.
5. Electrophoresis Buffer Series
Tae tsis (1 ×): Cov lus qhia DNA agarrough entrophoresis (cov kev daws teeb meem tsawg, tsim nyog rau cov ntawv loj DNA). TBE Tsis yog (1 ×), TBE Tsis (10 ×): Siab} Kev daws teeb meem DNA / RNA Electrophoresis (xws li cov thooj me lossis polyacrylamide); Ntev - sij hawm Electrophoresis (tsis muaj peev xwm ua tau zoo dua tae).
6. WESTERN BLOT TEXU NTSUAS FFFER Series
Western blup buffer (10 ×): ntas yeej siv los hloov cov protein los ntawm gel rau membrane (xws li duab thib ib), muaj tris, glycine thiab methanol.
Tris - hcol / l, ph6.8): npaj ntawm nplooj ntawv gel, thiab lwm yam.
Tris-HCl buffer (1.5mol/L, pH8.8): Preparation of SDS-PAGE separation gel, etc.
7. Txuj Ci P PH Buffer Series
Ph {{0} }/} / 6.86 / 9.18 Buffer (EG PH6.86 yog qhov nruab nrab, ph4.00 thiab 9.18 yog cov qauv acidic thiab alkaline).
Tsis yog reagents yog los ntawm tsis muaj txhais tau tias ib qho kev txhawb nqa kev txhawb nqa hauv chav kuaj. Lawv cov txiaj ntsig tseem ceeb nyob rau hauv kev muab cov chaw muaj txiaj ntsig thiab ruaj khov thiab ruaj khov rau kev tswj hwm lub thermodynamic equilibrium thiab tus nqi ntawm cov kev cuam tshuam zoo tshaj plaws. Los ntawm microenadironment kev cai ntawm enzyme cov chaw nquag, rau kev saib xyuas ntawm biomacroman yog qhov tseem ceeb tshaj plaws thiab kev ntseeg tau ntawm cov ntaub ntawv sim. Beekman yuav coj koj kev tshawb fawb txog kev tshawb fawb.
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