KAPILÁRNÍ ZÓNOVÁ ELEKTROFORÉZA CZE Capillaiy Electrophoresis Light source Computer Data in Cathode Data Out Chart Recorder E o co CM Time (min) Photocathode Buffer Anode HJERTEN 1967 1981 -JORGENSON LUKACSOVA CUN. CHEM. 27/9, 1551-1553 (1961) Free-Zone Electrophoresis in Glass Capillaries James W. Jorgenson and Krynn DeArman Lukacs A simple theory of zone electrophoresis in open-tubular capillaries is developed. According to this theory, to achieve the highest resolution of zones, tubes with as small an inside diameter as possible should be used in combination with as high an applied voltage as feasible. To test this approach, we performed electrophoresis In glass capillaries with an internal diameter of 75 fim and a length of 100 cm. A special fluorescence detector was used to detect fluorescent zones while they migrated inside the capillary. With the application of 30 kV potentials to this system, rapid and efficient separations of amino acids, peptides, and urinary amines were demonstrated. In all cases fluorescent derivatives were necessary for detection. Preliminary results are encouraging, and with further development of sensitive detection devices, applications in clinical analyses may be feasible. small diameter simultaneously facilitates heat transfer as well as stabilizes the medium. Regardless of the diameter, some radial temperature gradient will persist. However, this temperature gradient is undesirable only to the extent that a significant fraction of the solute molecules making up a zone spend longer than average times migrating within "cool" or "hot" regions of the tube radius. Here a more subtle effect of reduced tube diameters comes into play. If the diameter is Bmall enough that solute molecules diffuse back and forth across the tube many times during their migration, then the probability that a significant fraction of molecules will spend excessive amounts of time in any one portion of the tube radius is greatly reduced. Thus the solute molecules have an excellent chance of traveling throughout all portions of the tube radius, and any variations in migration velocity will tend to average out. To summarize, the possible advantages of performing zone electrophoresis in open tubes of small diameter are:_ A.B 10 15 Time (minutes ) ram of dansyl amino acids 20 BECKMAN 1987 2003 - PROJEKT LIDSKEHO GENOMU q Ciplllar; arrrp Eacti of sot s-ofi '. *■ capJianci Array wlnticw* Thoporr flctococ*i f.c jn hsod c-snfxaJ User ctjocwa Anote chamber imcr ntgrt proBurg Arm trwc cut Cathode stage : • 4 DMA unpes aro PROČ CE A BIOCHEMIE? VÝHODY CE ■ Aplikační diverzita nabité i neutrálni látky nízkomolekulární i vysokomolekulárni látky chirální i achirální látky bakterie i viry VÝHODY CE ■ Aplikační diverzita ■ Jednoduchá instrumentace VÝHODY CE > Aplikační diverzita > Jednoduchá instrumentace > Vysoké rozlišení a účinnost separací > Malá spotřeba vzorku > Rychlost analýzy > Malá spotřeba chemikálií a malé množství adu nm U NI SCI MÓDY CZE Use CE Mode.„ For Analysis of++. Capillary zone electrophoresis (CZE) Ions, etc* Mi cellar electro kinetic chromatography (MEKC) Neutral and ionic analytes Chiral capillary electrophoresis (CCE) Chiral molecules Capillary eleetrochromatography (CEC) Small molecules Capillary gel electrophoresis (CGE/SDS-PAGE) DNA/PNA size/protein MW Capillary isoelectric focussing (CIEF) Protein/peptide isoelectric point. Capillary isotachophoiesis (CITP) Ions KAPILÁRNI ZONOVA ELEKTROFORÉZA VE VOLNÉ KAPILÁŘE VÝSLEDNÁ MOBILITA ČÁSTIC PŘI CZE SEPARACE ANIONTŮ POMOCÍ U NI SCI ímAUj 40 n 30 20 10 NO, Criy Br I SCN- BrO, CIO, u AslV ~i— 10 Sami) l e 100 mg/l each anályte Buffer 20 iiiiVlphosphate,pH 80 Capillary DB WAX (JAW) i = r>fi cm L - tni iti = 50 UJR i Injection 200 lubar ♦ s Temperature 20 ůc 1 12 Time [min] Electric Field 2W V/cm, reversed polarity Defection Sign jíl 200,10 nm Reference 4"íOh 80 lun STANOVENÍ AKTIVITY HD POMOCÍ ■ U 1 u I 1 ■ 1 1 I 1 1} Fig. 1. CZE analysis of haloalkane dehalogenase catalysed reaction using indirect detection. Overlaid electropherograms show the reaction mixture of 10 raM 1.3-dibromopropane in 50 niM glycine buffer (pH 9.01 at 30°C before and Q. 13. 27. 36 and 45 min after addition of 10 jjlI of enzyme solution. The separation conditions are the same as in Table 1. Fig. 2. CZE analysis of haloalkane dehalogenase catalysed reaction using long-end injection in combination with direct detection. The enzyme preparation was 4 times diluted with 50 wii phosphate buffer (pH 7.5). Separation conditions ait the same as in Table 2. The other conditions are the same as in Fie. 1. MICELÁRNÍ ELEKTROKINETICKÁ CHROMATOGRAFIE PRINCIP MEKC 1 ■ MICELA 1 a - střed - rozpustná v obou b - silně hydrofilní - nerozpustná v micele c - silně hydrofóbní - nerozpustná ve vodné fázi SEPARACE FENOLŮ A ALKOHOLŮ POMOCÍ MEKC UNI SCI [mAU] 16 14-12 ID 8 6 4 5 6 8 10 11 12 14 13 7.5 10 1Z.5 15 17.5 IQ 22.5 25 25.5 1 4-hyriroxybenzyl alcohol (18 ppm) 6 2 3-hydroxybenzyl alcohol (9 ppmt 7 3 Phenol (26 ppm} 8 4 2-liydroHybenzyl alcohol (18 ppin) 9 5 m-cresol (19 ppm) Time [min] p-cresol(32 ppm) 2-chloropbenol 122 ppm) 216-xylenoH36 ppm) o-ettiylphenol (23 ppmf Buffer 90 mM borate, pH 8-6, 70 mM SI Capillary PVA-eoated (no EOF) L = 56 em L - 64.5 cm id = 50 ym BF 3 Injection 20 mbar ■ s Electric Field 465 V/cra Temperature Capillary 12 X Detection Signal 20W10 mu Reference 350/80 nm 10 2,3-nylenol (20 ppm) 11 2,5-xylenol (22ppm) 12 3,4-xylenol (23 ppm) 13 3,5-xylenol (19 ppm) 14 2r4-xylenol (27 ppm) STANOVENÍ AKTIVITY CYP 2C9 POMOCÍ MEKC m*U 16 14 11 Diclofenac 120 minutes SOmjnut^ 60 minutes —i - - —' 30 minutes 4-hydroxydiclofenac i » <—r—i—i t t i » » í i—r -I- 10 n—'—*—1—?—'—■—■—i—'—1—■—i—■— M m k u m Figure 3. MEKC analysis of CYP2C9 reaction. Overlaid elec-tropherograms show the enzymatic reaction under standard conditions as described in Section 2; the final concentrations of CYP2C9 and diclofenac in reaction mixture were 9.7 nM and 100uM respectively. MEKC conditions as in Fig. 2. KAPILÁRNÍ GELOVÁ ELEKTROFORÉZA CGE FRAGMENTÜ dsDNA I SCI Absorbance [mAU] 8 6 15 14 13 12 8 9 10 11 S § -To i5 iT IT log [bp] 3.2 3.0 2.8 2.6 2,4 2.2 2.0 1.8 36 bp 51 bp 65 bp 75 bp 126 bp 179 bp 7 222 bp 8 350 bp 9 396 bp 10 460 bp 11 517 bp 12 676 bp 13 1198 bp 14 1605 bp 15 2645 bp 1i5 flj iT Time [min] Ferguson Plot (calibration for size determination) 0.030 0.035 0.D4Q 0.045 0.050 Time [mil] Capillary: CEP Coated Capillary, 1/ L 40/18.5 cm, i.d. 75 um Sample: pGEM DNA Markers, 1 pgrpL Buffer: DNA Buffer +■ 1.5% Electrokinetic Injection: -5 kV, 4 s Voltage: -16,5 kV Temperature: 25 ■ C Detection: 260 run with DAD illter for 260 run (optional) INSTRUMENTACE CZE SCHEMA ZAŘÍZENI PRO CZE U PC control ▼ Outlet reservoir Power supply Capillary NAPÁJECÍ ZDROJ KAPILÁRA stabilizovaný ± 30 kV 300 |jA konstantní napětí nebo proud obojí polarita ochrana obsluhy křemenná - 25 -100 pm i.d - 350 |jm o.d délka 10 až 100 cm polyimidové vnější pokrytí DÁVKOVANÍ - HYDRODYNAMICKÉ DÁVKOVANÍ - ELEKTROKINETICKÉ ts = eel UV light source \7 0©n@e©0 ©^ e ®n@e©G0 ®e where £ = molar absorptivit c = concentration / = pathlength UNI SCI lime DETEKCE FLUORESCENČNÍ 2003 - PROJEKT LIDSKÉHO GENOMU 3730XL DNAANALYZER APPLIED BIOSYSTEMS 3730x1 DNA Analyzer 84 KLASICKÁ CZE MICROCHIP CZE 3 < O C CO -Q s_ O to -Q < 0.0 ■1.0 " A Cr - r* - ľ" ■ EDTA ......... 2 4 6 8 10 12 35 30 < E 25 02 + 4H30+ + 4e- OH- 4H20^-2H2 + 40H--4e- Ampholyty u r j i •CH2 —N-(CH2)n-N-CH2- (CH2)n R NR3 R-CH3 - CH2COOH - H IZOELEKTRICKÁ FOKUSACE IZOELEKTRICKÁ FOKUSACE KAPILÁRNÍ IZOELEKTRICKÁ FOKUSACE IZOELEKTRICKÁ FOKUSACE ANALYTICKÁ ■ Provedení - v gelech - PAGE, agarosa Použití - sledování komplexních směsí U SCI I - izoenzymové složení - stanovení pl - rozřezání a eluce - LipH elektrody - pl standardy IZOELEKTRICKÁ FOKUSACE ANALYTICKÁ IZOELEKTRICKA FOKUSACE ANALYTICKA - STANDARDY ■Cytochrome c-1— = Lenlil lectin Human hemoglonin C-^ Human hemoglobin AfOx^ Equine myoglobin Equine myoglobin minor nand Human carbonic anh^Jrase - Bowie catxxncartrydra.se |j-Lactoqlobulin B i Phycocyan in- B \lEF Standards for accurate pi calibration of naftye tEF qols. A 5 ui of th& standards ivera sfaihsd Li'j'm Coomasste bius R-250 dye and \c,foc&in seartet. fiL S^jict Xfto standards wore un&talrtod. IZOELEKTRICKÁ FOKUSACE IMOBILINE IZOELEKTRICKÁ FOKUSACE PREPARATIVNÍ ill 4 IZOELEKTRICKÁ FOKUSACE PREPARATIVNÍ ■ Provedení - v sypaných vrstvách (Sephadex) - v gradientech hustoty (sacharóza) Použití - izolace bílkovin ľ 1U IJI SCI DVOUROZMĚRNÉ METODY ■ Metoda titračních křivek ■ Dvourozměrná elektroforéza METODA TITRAČNÍCH KŘIVEK METODA TITRACNICH KŘIVEK JU U METODA TITRAČNÍCH KŘIVEK DVOJROZMĚRNÁ ELEKTROFORÉZA Equilibration Protein extract i SDS-PAGE DVOJROZMERNÁ ELEKTROFORÉZA 1975 OTARRELL DVOJROZMĚRNÁ ELEKTROFORÉZA *l ^ * 'A +? Uj Lj Uj 4jI> \—\-1-1-h-h tů.ooo 66,200 ■§, 43.000 ^ 36,ooo , i— r ai.aaa 21.50.0. 17,500 - 6 1-h rivQ-drrTiensforTaJ eJectincp beret re I pattern of trhe £-D SQ5-PdG£ Standards separated on the Mini-PROTEAN ft ceil. 2.5 ware applied to a 3.S% tube gei crosstinked with pipara2sne diacryiarfiide containing rVT urea and 2% B\o-Lyte ampholytes (t part 3/1QT 2 parts 5/7). The tube gel ivss run ori a crossiinked with bis and stiver stained. For details, see references 21, 22, and 23. DVOJROZMĚRNÁ ELEKTROFORÉZA Pí Základní schéma analýzy užívané v proteomice Směs proteinu 2. Izolace Štěpení trypsinem 4. Sekvenční analýza Fragmentace peptidů Q Peptidy j ----------- Sekvence peptidu 3. Hmotnostní analýza Hmotnostní spekrometrie /límot motnostní spektra" peptidů Cm»MornZ3 0ctfcIop v Ml Btia t r-* n E*i £<* |HM0e Spoít Aign AŕtafeM Utofe» L*0 IW>_ i «rim^i «ami till maial a-iiii n i ebibibisi míE bwisibi mala J LI 2 - i-.** Ii lů 1 Xl| Yl| REH «I «t?l *4Ľ 101t M 8031 nv *i 11053 Ali 0 C MB 295 45 3712 250 51 3745 155 COOS MB 242 48 ľ208 237 S3 2521 3575 0 275 MM 11» 4Í M 125 53 107$ 2480 0553 10» 100 50 3230 108 54 4738 7240 0 381 Ii»? 1« » MÔ4 1» ö »5 WÍ ÖW 1029 55 52 1150 MM 221 52 5403 217 55 44% 4555 0184 10» 284 52 MM MM 275 53 MM MM 147 S3 3078 151 55 1281 8685 0825 10« 290 58 10« 35 57 1005 127 2nd 184] Ji DIGE - DIFERENČNÍ GELOVÁ ELEKTRO FORESA Cy2 i excitace 488 nm emise 520 nm 400 500 600 800 pooled sample - internal standard Cy2 label image of Cy2 internal atorvdord healthy Cv3 label 4- cor I i ■: . r mix*u r© IPG 'otn conjugate slreptavidin horseradiäh poroxidasa complex primáry anlibod 125l-protein A enzymem značená sekundární protilátka - konjugace s peroxidasou (tetrazoliová sůl), alkalickou fosfatasou zlatem značená sekundární protilátka (100 pg) chemiluminiscence - nejcitlivější DETEKCE U -i- ft V Bbcking pgcnr J DETEKCE SPECIÁLNÍ APLIKACE BLOTTINGU ■ Získání homogenních preparátů ■ Studium složení a struktury bílkovin ■MS bílkovin-MALDI, DESI ■ Příprava protilátek ■ Purifikace protilátek MU NI SCI DETEKCE DETEKCE