{"id":2659,"date":"2021-04-12T12:06:23","date_gmt":"2021-04-12T10:06:23","guid":{"rendered":"https:\/\/www.fractioncollector.info\/?p=2659"},"modified":"2023-07-26T07:52:57","modified_gmt":"2023-07-26T05:52:57","slug":"purifikace-proteinu-kapalinova-chromatografie-gradientova-eluce","status":"publish","type":"post","link":"https:\/\/www.fractioncollector.info\/de\/purifikace-proteinu-kapalinova-chromatografie-gradientova-eluce\/","title":{"rendered":"\u00dasp\u011b\u0161n\u00e1 purifikace protein\u016f: gradientov\u00e1 eluce"},"content":{"rendered":"

Purifikace protein\u016f: kapalinov\u00e1 chromatografie & gradientov\u00e1 eluce<\/span><\/h1>\n

s kolektorem frakc\u00ed OMNICOLL a LAMBDA peristatick\u00fdmi pumpami<\/span><\/h2>\n<\/div><\/div><\/div><\/div><\/div>

Pro \u010di\u0161t\u011bn\u00ed protein\u016f<\/strong> a izolaci peptid\u016f, nukleov\u00fdch kyselin nebo mal\u00fdch molekul<\/strong> ze sm\u011bsi fermenta\u010dn\u00edho media se pou\u017e\u00edv\u00e1 kapalinov\u00e1 chromatografie (LC) s<\/strong> iontovou v\u00fdm\u011bnou<\/strong>, hydrofobn\u00ed interakc\u00ed, adsorpc\u00ed nebo s vylou\u010den\u00edm podle velikosti molekul.<\/p>\n

Za \u00fa\u010delem optimalizace proces\u016f kapalinov\u00e9 chromatografie<\/strong> se v laboratorn\u00ed praxi \u00fasp\u011b\u0161n\u011b pou\u017e\u00edvaj\u00ed sb\u011bra\u010de frakc\u00ed OMNICOLL a spolehliv\u00e1 peristaltick\u00e1 \u010derpadla LAMBDA:<\/p>\n<\/div><\/div><\/div><\/div>

P\u0159\u00edklad uspo\u0159\u00e1d\u00e1n\u00ed s automatick\u00fdm sb\u011bra\u010dem frakc\u00ed<\/span><\/h3>\n

Nap\u0159\u00edklad kompletn\u00ed uspo\u0159\u00e1d\u00e1n\u00ed kolonov\u00e9 chromatografie s automatick\u00fdm sb\u011brem<\/strong> elu\u00e1tu pomoc\u00ed sb\u011bra\u010de frakc\u00ed OMNICOLL zahrnuje:<\/p>\n

\"Sb\u011bra\u010d<\/p>\n

    \n
  • Dva obdobn\u00e9 z\u00e1sobn\u00edky s elu\u010dn\u00edm pufrem A a B<\/strong> spojen\u00e9 n\u00e1soskou<\/strong> (nebo trubkou ve spodn\u00ed \u010d\u00e1sti s ventilem pro nastaven\u00ed pr\u016ftoku)<\/li>\n
  • Elu\u010dn\u00ed pufr A<\/strong> je trvale nas\u00e1v\u00e1n do k\u00e1dinky s elu\u010dn\u00edm pufrem B,<\/strong> kter\u00fd je neust\u00e1le m\u00edch\u00e1n magnetick\u00fdm m\u00edchadlem<\/strong>, \u010d\u00edm\u017e se m\u011bn\u00ed jeho koncentrace<\/li>\n
  • Programovateln\u00e1 peristaltick\u00e1 pumpa LAMBDA pumpuje prom\u011bnlivou sm\u011bs<\/strong> do kolony<\/li>\n
  • Elu\u00e1t z kolony<\/strong> proch\u00e1z\u00ed detektorem<\/strong> (m\u011b\u0159en\u00ed pH, absorbance p\u0159i 280 nm, vodivosti)<\/li>\n
  • From the detector, the protein fractionation<\/b> is collected by the OMNICOLL fraction collector<\/b> into the desired containers (e.g.: tubes, vials, etc.)<\/li>\n
  • Z detektoru jsou frakce<\/strong> sb\u00edr\u00e1ny sb\u011bra\u010dem frakc\u00ed OMNICOLL<\/strong> do po\u017eadovan\u00fdch n\u00e1dob (nap\u0159.: zkumavky, lahvi\u010dky atd.)<\/li>\n<\/ul>\n

    Proteiny se eluuj\u00ed zm\u011bnou slo\u017een\u00ed pufru (iontov\u00e1 s\u00edla, pH, vodivost, koncentrace sol\u00ed atd.). Elu\u010dn\u00ed podm\u00ednky lze m\u011bnit zm\u011bnou toku pufru A do B, ve kter\u00e9m se slo\u017een\u00ed mobiln\u00ed f\u00e1ze v pr\u016fb\u011bhu \u010dasu m\u011bn\u00ed.<\/p>\n<\/div><\/div><\/div><\/div>

    Postup<\/h2>\n

    P\u0159i kapalinov\u00e9 chromatografii je d\u016fle\u017eit\u00e9 m\u00edt kolonu napln\u011bnou stacion\u00e1rn\u00ed f\u00e1z\u00ed rovnom\u011brn\u011b bez vzduchov\u00fdch bublin, kan\u00e1l\u016f a trhlin.<\/strong><\/p>\n

    Peristaltick\u00e1 pumpa LAMBDA \u010derp\u00e1 kapalnou mobiln\u00ed f\u00e1zi kontinu\u00e1ln\u011b<\/strong> do napln\u011bn\u00e9 chromatografick\u00e9 kolony konzistentn\u00edm reprodukovateln\u00fdm pr\u016ftokem bez pulzac\u00ed.<\/strong><\/p>\n

    Slo\u017eky fermenta\u010dn\u00edho m\u00e9dia jsou adsorbov\u00e1ny a desorpov\u00e1ny v r\u016fzn\u00e9m rozsahu na stacion\u00e1rn\u00ed f\u00e1zi, zat\u00edmco slo\u017eky v mobiln\u00ed f\u00e1zi se od sebe pomalu odd\u011bluj\u00ed.<\/strong><\/p>\n

    Mobiln\u00ed f\u00e1ze prot\u00e9k\u00e1 kolonou a odchoz\u00ed elu\u00e1t na spodn\u00edm konci chromatografick\u00e9 kolony je sb\u00edr\u00e1n pomoc\u00ed sb\u011bra\u010de frakc\u00ed OMNICOLL.<\/strong><\/p>\n

    Detektor, kter\u00fd kontinu\u00e1ln\u011b sleduje eluci proteinu z kolony nebo anal\u00fdzu frakc\u00ed, poskytuje informace o koncentraci po\u017eadovan\u00e9ho proteinu ve frakci pro dal\u0161\u00ed zpracov\u00e1n\u00ed, nap\u0159\u00edklad: gelovou elektrofor\u00e9zou.<\/p>\n

    \n<\/div><\/div><\/div><\/div>

    Optimalizace elu\u010dn\u00edch metod p\u0159i purifikaci protein\u016f<\/h2>\n

    V\u00fdb\u011br spr\u00e1vn\u00e9ho elu\u010dn\u00edho pufru m\u016f\u017ee zna\u010dn\u011b zlep\u0161it kvalitu separace a \u010das anal\u00fdzy p\u0159i purifikaci enzym\u016f.<\/p>\n<\/div><\/div><\/div><\/div>

    Izokratick\u00e1 eluce v kapalinov\u00e9 sloupcov\u00e9 chromatografii<\/h3>\n

    V kapalinov\u00e9 chromatografii jsou proteiny odd\u011bleny za konstantn\u00edho slo\u017een\u00ed mobiln\u00ed f\u00e1ze (tj. izokratick\u00e1 eluce):<\/p>\n

      \n
    • Pokud vzorky nejsou p\u0159\u00edli\u0161 slo\u017eit\u00e9 a tak\u00e9 kdy\u017e vzorky nemaj\u00ed podobn\u00e9 reten\u010dn\u00ed \u010dasy.<\/li>\n<\/ul>\n

      Krom\u011b toho u nejpevn\u011bji adsorbovan\u00fdch protein\u016f bude eluce s konstantn\u00edm slo\u017een\u00edm mobiln\u00ed f\u00e1ze trvat velmi dlouho.<\/strong><\/p>\n

      P\u0159i izokratick\u00e9 eluci se pou\u017e\u00edv\u00e1 pufr a peristaltick\u00e1 pumpa.<\/p>\n<\/div><\/div><\/div>

      Gradientov\u00e1 eluce \u2013 pro purifikaci protein\u016f z komplexn\u00edch sm\u011bs\u00ed (biologick\u00e9 vzorky)<\/h3>\n

      P\u0159i gradientov\u00e9 eluci se slo\u017een\u00ed mobiln\u00ed f\u00e1ze m\u011bn\u00ed b\u011bhem separace a ovliv\u0148uje retenci analyt\u016f.<\/p>\n

      Gradientov\u00e1 eluce se pou\u017e\u00edv\u00e1 p\u0159i purifikaci protein\u016f:<\/p>\n

        \n
      • Slo\u017eit\u00e9 biologick\u00e9 vzorky<\/strong><\/li>\n
      • Sm\u011bsi se zn\u00e1m\u00fdmi vlastnostmi<\/strong><\/li>\n
      • Komponenty s podobn\u00fdmi reten\u010dn\u00edmi \u010dasy<\/strong><\/li>\n<\/ul>\n

        Pokud se k purifikaci protein\u016f v kapalinov\u00e9 kolonov\u00e9 chromatografii pou\u017e\u00edv\u00e1 gradientov\u00e1 eluce, m\u016f\u017ee se reten\u010dn\u00ed \u010das zkr\u00e1tit.<\/strong><\/p>\n

        Gradientov\u00e1 eluce se prov\u00e1d\u00ed se dv\u011bma pufry<\/strong> a s jednou nebo dv\u011bma peristaltick\u00fdmi pumpami.<\/strong><\/p>\n<\/div><\/div><\/div><\/div><\/div>

        Gradientov\u00e1 eluce a elu\u010dn\u00ed profil<\/h3>\n

        P\u0159i\u00a0gradientov\u00e9 eluci\u00a0se m\u011bn\u00ed s\u00a0\u010dasem slo\u017een\u00ed mobiln\u00ed f\u00e1ze pro optim\u00e1ln\u00ed separaci vzorku.<\/p>\n

        Reten\u010dn\u00ed faktor a t\u00edm i kapacitn\u00ed faktor mohou b\u00fdt ovlivn\u011bny odli\u0161n\u00fdm slo\u017een\u00edm eluentu (gradientov\u00e1 eluce).<\/p>\n

        Profily gradientu:<\/p>\n

        \"line\u00e1rn\u00ed \"\"\u00a0\"krokov\u00e1<\/p>\n

        Neline\u00e1rn\u00ed gradienty nab\u00edzej\u00ed n\u011bkolik odli\u0161n\u00fdch v\u00fdhod, nap\u0159\u00edklad krat\u0161\u00ed doba separace, lep\u0161\u00ed rozli\u0161en\u00ed vzorku anebo vy\u0161\u0161\u00ed citlivosti detekce.<\/strong><\/p>\n<\/div><\/div><\/div><\/div>

        \"peristaltick\u00e1<\/p>\n

         <\/p>\n

         <\/p>\n

         <\/p>\n

        Biochemici doporu\u010duj\u00ed peristaltick\u00e9 pumpy LAMBDA, kter\u00e1 zaru\u010duj\u00ed aplikaci mobiln\u00ed f\u00e1ze bez pulzac\u00ed.<\/em><\/p>\n<\/div><\/div><\/div><\/div><\/div>

        Generov\u00e1n\u00ed gradientu spolehliv\u00fdmi peristaltick\u00fdmi pumpami<\/h3>\n

        Laboratorn\u00ed peristaltick\u00e9 pumpy LAMBDA jsou programovateln\u00e9 a d\u00e1lkov\u011b ovladateln\u00e9 pro dosa\u017een\u00ed line\u00e1rn\u00edho, krokov\u00e9ho gradientu.<\/p>\n<\/div><\/div><\/div><\/div>

        Gradientov\u00e1 eluce s jednou pumpou<\/span><\/h3>\n

        Gradientovou eluci lze prov\u00e1d\u011bt tak\u00e9 s jednou peristaltickou pumpou: n\u00e1doby s elu\u010dn\u00edm pufrem A & B jsou spojeny sifonem nebo trubkou s ventilem pro nastaven\u00ed pr\u016ftoku.<\/p>\n

          \n
        1. Elu\u010dn\u00ed pufr A je permanentn\u011b nata\u017een do k\u00e1dinky s dob\u0159e prom\u00edchan\u00fdm elu\u010dn\u00edm pufrem B, kde se m\u011bn\u00ed koncentrace pufru.<\/li>\n
        2. Programovateln\u00e1 peristaltick\u00e1 pumpa LAMBDA je pou\u017eita k \u010derp\u00e1n\u00ed sm\u011bsi do chromatografick\u00e9 kolony.<\/li>\n
        3. Elu\u00e1t z kolony d\u00e1le proch\u00e1z\u00ed detektorem pro zji\u0161t\u011bn\u00ed pH, absorbance p\u0159i 280 nm a vodivosti.<\/li>\n
        4. Z detektoru se frakce postupn\u011b j\u00edmaj\u00ed sb\u011bra\u010dem OMNICOLL do po\u017eadovan\u00fdch n\u00e1dob (nap\u0159.: lahve, k\u00e1dinky, zkumavky atd.).<\/em><\/li>\n<\/ol>\n<\/div><\/div><\/div>

          Gradientov\u00e1 eluce s dv\u011bma pumpami<\/span><\/h3>\n

          Dv\u011b programovateln\u00e9 peristaltick\u00e9 pumpy LAMBDA<\/strong>\u00a0 jsou pou\u017eity pro reprodukovateln\u00e9 m\u00edch\u00e1n\u00ed eluent\u016f pro gradientovou eluci.<\/p>\n

            \n
          1. Zm\u011bnu slo\u017een\u00ed v pr\u016fb\u011bhu \u010dasu lze uskute\u010dnit pomoc\u00ed zad\u00e1n\u00ed programu p\u0159\u00edmo na displeji peristaltick\u00e9 pumpy LAMBDA nebo ji lze zaznamenat a zobrazit jako graf v re\u00e1ln\u00e9m \u010dase pomoc\u00ed ovl\u00e1dac\u00edho programu PNet.<\/li>\n
          2. Gradient sm\u011bsi proch\u00e1z\u00ed p\u0159es chromatografickou kolonou a slo\u017eky jsou separov\u00e1ny.<\/li>\n
          3. Elu\u00e1t z kolony d\u00e1le proch\u00e1z\u00ed detektorem pro zji\u0161t\u011bn\u00ed pH, absorbance p\u0159i 280 nm a vodivosti.<\/li>\n
          4. Automatick\u00fd sb\u011bra\u010d frakc\u00ed LAMBDA OMNICOLL, frakcionuje elu\u00e1t a sb\u00edr\u00e1 frakce na z\u00e1klad\u011b naprogramovan\u00e9ho \u010dasu nebo po\u010d\u00edta\u010de kapek.<\/li>\n<\/ol>\n<\/div><\/div><\/div><\/div>
            \n

            \u00a0<\/p>\n<\/div><\/div>

            Po\u017eadavky na \u010derpac\u00ed syst\u00e9m pro sloupcovou chromatografii<\/span><\/h2>\n

             <\/p>\n<\/div><\/div><\/div><\/div>

            Nejd\u016fle\u017eit\u011bj\u0161\u00ed po\u017eadavky na \u010derpac\u00ed syst\u00e9m:<\/p>\n

              \n
            • Konstantn\u00ed a reprodukovateln\u00fd pr\u016ftok<\/strong> <\/li>\n<\/ul>\n

              Reprodukovatelnost z\u00e1vis\u00ed na reten\u010dn\u00edch \u010dasech p\u00edk\u016f. Reten\u010dn\u00ed \u010dasy jsou z\u00e1visl\u00e9 na pr\u016ftoku.<\/p>\n

                \n
              • Pr\u016ftok bez pulzac\u00ed<\/strong><\/li>\n<\/ul>\n

                Stacion\u00e1rn\u00ed f\u00e1ze mus\u00ed b\u00fdt chr\u00e1n\u011bna proti tlakov\u00fdm n\u00e1raz\u016fm. Z\u00e1kladn\u00ed linie detektoru by m\u011bla z\u016fstat stabiln\u00ed.<\/p>\n

                \n<\/p>

                LAMBDA MULTIFLOW programovateln\u00e9 peristaltick\u00e9 pumpy jsou vhodn\u00e9 k dosa\u017een\u00ed reprodukovateln\u00fdch pr\u016ftok\u016f a maj\u00ed 5letou z\u00e1ru\u010dn\u00ed dobu.<\/p>\n<\/div><\/div><\/div>

                \"Programovateln\u00e1<\/p>\n<\/div><\/div><\/div><\/div>

                Po\u017eadavky sb\u011bru p\u0159i purifikaci protein\u016f<\/span><\/h2>\n

                 <\/p>\n<\/div><\/div><\/div><\/div>

                Nejd\u016fle\u017eit\u011bj\u0161\u00ed po\u017eadavky sb\u011bra\u010de frakc\u00ed pro kapalinovou sloupcovou chromatografii jsou:<\/p>\n

                  \n
                • Neomezen\u00e1 kapacita<\/strong><\/li>\n<\/ul>\n

                  Mo\u017enost sb\u00edrat neomezen\u00fd po\u010det frakc\u00ed je nesm\u00edrn\u011b d\u016fle\u017eit\u00e1, zejm\u00e9na p\u0159i optimalizaci procesu.<\/p>\n

                    \n
                  • \n

                    Rozsah objemu<\/strong><\/p>\n<\/li>\n<\/ul>\n

                    Mo\u017enost p\u0159izp\u016fsobit sb\u011bra\u010d frakc\u00ed pro r\u016fzn\u00e9 laboratorn\u00ed experimenty a procesy v pilotn\u00edm f\u00e1zi.<\/p>\n

                    P\u0159izp\u016fsobiteln\u00fd sb\u011bra\u010d frakc\u00ed OMNICOLL je ide\u00e1ln\u00ed pro efektivn\u00ed optimalizaci proces\u016f v laboratorn\u00edm a pilotn\u00edm m\u011b\u0159\u00edtku.<\/p>\n<\/div><\/div><\/div>

                    \"Sb\u011bra\u010d<\/p>\n<\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"

                    Purifikace protein\u016f: kapalinov\u00e1 chromatografie & gradientov\u00e1 eluce s kolektorem frakc\u00ed OMNICOLL a LAMBDA peristatick\u00fdmi pumpami Pro \u010di\u0161t\u011bn\u00ed protein\u016f a izolaci peptid\u016f, nukleov\u00fdch kyselin nebo mal\u00fdch molekul ze sm\u011bsi fermenta\u010dn\u00edho media se pou\u017e\u00edv\u00e1 kapalinov\u00e1 chromatografie (LC) s iontovou v\u00fdm\u011bnou, hydrofobn\u00ed interakc\u00ed, adsorpc\u00ed nebo s vylou\u010den\u00edm podle velikosti molekul. Za \u00fa\u010delem optimalizace proces\u016f kapalinov\u00e9 chromatografie se v […]<\/p>\n","protected":false},"author":2,"featured_media":2638,"comment_status":"closed","ping_status":"closed","sticky":true,"template":"","format":"standard","meta":{"footnotes":""},"categories":[307],"tags":[],"class_list":["post-2659","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-uncategorized-cs","post-with-thumbnail","post-with-thumbnail-icon"],"yoast_head":"\n\u00dasp\u011b\u0161n\u00e1 purifikace protein\u016f: gradientov\u00e1 eluce – LAMBDA OMNICOLL | LAMBDA Laboratory Instruments<\/title>\n<meta name=\"description\" content=\"D\u016fle\u017eit\u00e9 aspekty, kter\u00e9 je t\u0159eba vz\u00edt v \u00favahu p\u0159i v\u00fdb\u011bru peristaltick\u00fdch pump a sb\u011bra\u010de frakc\u00ed p\u0159i gradientov\u00e9 eluci v kapalinov\u00e9 kolonov\u00e9 chromatografii.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, 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