Chromatography yog ib txoj hauv kev los cais cov tshuaj. Nws yog siv rau kev soj ntsuam zoo thiab ntau yam ntawm lub cev thiab tshuaj lom neeg ntawm microparticles. Ib tug variation ntawm no technology yog affinity chromatography. Lub tswv yim ntawm kev sib txawv ntawm cov protein sib txuas siv cov cuab yeej ntawm molecular affinity tau paub hauv kev tshawb fawb rau ntau xyoo lawm. Txawm li cas los xij, nws tau txais nws txoj kev loj hlob tsuas yog nyob rau xyoo tas los no, tom qab kev qhia txog cov khoom siv hydrophilic ntxeem tau siv los ua matrix. Txoj kev no tso cai rau kev daws teeb meem kev tshuaj xyuas ob qho tib si (kev sib cais ntawm cov tshuaj thiab lawv qhov kev txheeb xyuas) thiab teeb meem kev npaj (purification, concentration).
Essence
Affinity chromatography (los ntawm lo lus Latin affinis - "nyob ib sab", "koom nrog") yog raws li kev sib cuam tshuam, uas yog tsim los ntawm kev sib txuas tshwj xeeb ntawm ib qho chaw molecule (ligand lossis affinant) thiab lub hom phiaj molecule. Cov txheej txheem no nthuav dav hauv qhov xwm txheej (kev sib txuas ntawm cov neeg nruab nrab lossis cov tshuaj hormones thiab receptors, cov tshuaj tiv thaiv thiabantigens, hybridization ntawm polynucleotides thiab lwm yam txheej txheem). Hauv tshuaj, affinity chromatography tau siv rau lub hom phiaj siv txij li xyoo 1951
Cov khoom sib cais raws li hauv qab no:
- ua haujlwm cov tshuaj uas muaj cov tshuaj kom cais tawm yog dhau los ntawm sorbent;
- ligand tso rau ntawm sorbent matrix khaws cov tshuaj no;
- nws yog concentrated (sau);
- rho tawm cov khoom cais tawm ntawm cov sorbent los ntawm kev ntxuav nrog cov kuab tshuaj.
Txoj kev no tso cai rau koj cais tag nrho cov hlwb. Qhov txawv ntawm cov tsoos sorption chromatography yog qhov muaj zog biospecific khi ntawm cov khoom sib cais rau cov sorbent, uas yog tus cwj pwm los ntawm kev xaiv siab.
Adsorbents
Cov tshuaj hauv qab no yog siv los ua adsorbents:
- Gel compounds raws li agarose, polysaccharide tau los ntawm agar. Feem ntau siv yog 3 hom: sepharose 4B, CL (cross-linked agarose) thiab affi-gel. Cov txheej txheem tom kawg yog hloov kho gel ntawm agarose thiab polyacrylamide. Nws muaj ntau dua lom inertness, siab tshuaj thiab thermal kuj.
- YSilica (silica gel).
- iav.
- YOrganic polymers.
Txhawm rau tshem tawm txhua yam teeb meem thaum sib cuag ligand, cov tshuaj ntxiv yog siv los cais nws ntawm cov cab kuj (peptides, diamines, polyamines, oligosaccharides).
khoom siv
Affinity chromatography cov cuab yeej suav nrog cov hauv qab no:
- tso tsheb hlau luam rau theem mobile (eluent);
- siab twj tso kua mis rau nruab nrab mov (feem ntau reciprocating);
- filter rau ntxuav eluents los ntawm hmoov av;
- khoom siv;
- chromatographic kem rau kev sib cais sib cais;
- ntes kom pom cov khoom sib cais tawm hauv kab;
- chromatogram recorders thiab microprocessor unit (lub computer).
Txhawm rau txo cov huab cua yaj, helium yog thawj zaug dhau los ntawm theem txawb. Txhawm rau hloov qhov concentration ntawm eluent, ntau lub twj tso kua mis tswj los ntawm tus programmer raug teeb tsa. Cov kab chromatographic yog ua los ntawm cov hlau tsis muaj steel (rau cov kev xav tau ntxiv rau kev tiv thaiv corrosion), iav (kev xaiv universal) lossis acrylic. Rau kev npaj, lawv txoj kab uas hla tuaj yeem sib txawv ntawm 2 txog 70 cm. Hauv kev txheeb xyuas chromatography, microcolumns Ø10-150 µm yog siv.
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Methodology
Muaj 2 yam tseem ceeb ntawm cov kua affinity chromatography:
- Kem, nyob rau hauv uas kem yog sau nrog ib tug nyob twj ywm theem thiab ib tug sib tov yog dhau los ntawm nws nrog ib tug ntwseluent. Kev sib cais tuaj yeem tshwm sim hauv lub siab lossis hauv qab lub ntiajteb txawj nqus.
- txheej nyias. Lub eluent txav raws lub tiaj tus adsorbent txheej nyob rau hauv lub zog ntawm capillary rog. Cov adsorbent yog siv rau iav phaj, ceramic lossis quartz pas nrig, hlau ntawv.
theem tseem ceeb ntawm kev ua haujlwm suav nrog:
- npaj cov adsorbent, fixation ntawm ligand ntawm cov cab kuj;
- pub mis sib cais sib cais rau kab chromatographic;
- mobile theem loading, tivthaiv khi los ntawm ligand;
- phase hloov kom cais cov khoom khi.
Destination
Affinity chromatography yog siv los cais cov hom tshuaj hauv qab no (hom ligand siv tau qhia hauv cov kab tuav):
- analogues ntawm enzymatic inhibitors, substrates thiab cofactors (enzymes);
- bioorganic tshuaj nrog cov cim qhia ntawm caj ces alienness, kab mob thiab hlwb (antibodies);
- siab molecular hnyav carbohydrates, monosaccharide polymers, glycoproteins (lectins);
- nuclear proteins, nucleotidyltransferases (nucleic acids);
- receptors, thauj cov proteins (vitamins, hormones);
- proteins cuam tshuam nrog cell membranes (cells).
Cov thev naus laus zis no kuj tseem siv tau kom tau txais cov enzymes immobilized, thiab khi lawv rau cellulose tso cai rau tsim cov tshuaj tiv thaiv kab mob.
Chromatography ntawm DNA-binding proteins
Kev cais cov DNA-binding proteins yog ua los ntawm kev sivheparin. Qhov no glycosaminoglycan muaj peev xwm los khi ntau hom molecules. Affinity chromatography ntawm cov proteins ntawm pab pawg no yog siv los cais cov tshuaj xws li:
- yam tseem ceeb ntawm kev pib thiab elongation ntawm kev txhais lus (synthesis ntawm nucleic acid molecules thiab proteins);
- txwv tsis pub (cov enzymes uas paub txog qee yam kab ke hauv DNA ob leeg);
- DNA ligases thiab polymerases (enzymes uas ua kom muaj kev sib koom ua ke ntawm ob lub molecules los tsim cov tshuaj tshiab thiab koom nrog DNA replication);
- serine protease inhibitors uas ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob thiab kab mob;
- loj hlob yam: fibroblast, Schwann, endothelial;
- proteins ntawm extracellular matrix;
- hormone receptors;
- Ylipoproteins.
Nyob zoo ib tsoom niam txiv kwv tij neej tsas sawv daws.
Txoj kev no yog ib qho tshwj xeeb tshaj plaws rau kev sib cais ntawm cov tshuaj tiv thaiv kab mob (enzymes thiab cov kab mob loj dua - kab mob). Txawm li cas los xij, nws tsis yog siv los cais cov tshuaj lom neeg lom neeg xwb.
Kev kuaj xyuas cov tshuaj tiv thaiv hauv qhov me me, kev ntsuas ntau ntawm polyadenylic acid, kev txiav txim siab sai ntawm cov molecular pawg ntawm dehydrogenases, tshem tawm qee yam kuab paug, kawm txog kinetics ntawm kev ua kom tsis muaj zog ntawm trypsin, cov qauv molecular ntawm tib neeg. interferons - qhov no tsis yog tag nrho cov npe ntawm cov kev tshawb fawb uas siv affinity chromatography. Kev siv hauv tsev kho mob yog vim nws qhov zoo xws li:
- muaj peev xwm ntxuav tau zooproteins, polysaccharides, nucleic acids. Lawv txawv me ntsis ntawm lawv lub cev thiab tshuaj lom neeg thiab poob kev ua si thaum lub sij hawm hydrolysis, denaturation thiab lwm yam kev kho mob siv nyob rau hauv lwm txoj kev.
- Kev nrawm ntawm kev sib cais ntawm cov tshuaj, qhov xwm txheej zoo ntawm cov txheej txheem.
- Tsis xav tau tshwj xeeb enzyme purification thiab isoenzyme homogenization los txiav txim siab qhov sib txawv.
- Muaj peev xwm cais ntau yam khoom.
- Kev noj tsawg ntawm ligands.
- Muaj peev xwm sib cais ntawm cov khoom hauv ntim loj.
- Cov txheej txheem thim rov qab ntawm kev khi cov roj ntsha macromolecules.
Cov txheej txheem no tuaj yeem ua ke nrog lwm tus, txhawm rau txhawm rau txhawm rau ntxiv (gravitational, electromagnetic). Qhov no tso cai rau koj los nthuav cov peev xwm ntawm chromatography.
Enzymatic engineering
Ua tsaug rau txoj kev no, kev txhim kho nquag ntawm ceg tshiab ntawm biotechnology - enzyme engineering pib.
Affinity chromatography rau kev sib cais enzyme muaj qhov zoo hauv qab no:
- tau txais cov enzymes ntau ntau vim tias lub sijhawm tsawg, vim li ntawd - lawv txo nqi;
- immobilization ntawm enzymes tuaj yeem nthuav dav ntawm lawv daim ntawv thov hauv tshuaj thiab kev lag luam;
- Lub koom haum ntawm cov enzymes nrog cov khoom tsis sib haum xeeb ua rau nws tuaj yeem kawm txog kev cuam tshuam ntawm microenvironment thiab cov kev taw qhia ntawm cov tshuaj tiv thaiv, uas ua lub luag haujlwm tseem ceeb hauv cov txheej txheem ntuj tsim thiab lub cev.
