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ELVÁLASZTÁSTECHNIKAI MÓDSZEREK ELMÉLETE ÉS
GYAKORLATA Dr. Kremmer
Tibor VIII. AFFINITÁSI KROMATOGRÁFIA AFFINITY
CHROMATOGRAPHY (AFF) EÖTVÖS LORÁND
TUDOMÁNY EGYETEM Budapest 2013

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AFFINITÁSI KROMATOGRÁFIA
ALAPELV Kovalens kötéssel (általában köztes
láncon keresztül - spacer, leash, tentacle)
hordozó szemcsékre (mátrix) rögzített különbözo
funkcionális csoportok (ligandum), amelyek Az
elválasztandó (bio)molekulával specifikus és
reverzibilis kölcsönhatásokat képeznek. A
hordozó szemcsék anyaga (mátrix)
lehet - Poliszacharidok agaróz       
             (Sepharose, Superose)
cellulóz - Organikus polimerek poliakrilamid
hidroxialkil metakrilát (Spheron) etilénglik
ol metakrilát (Fractogel) - Kevert fázisok
(agaróz-poliakrilamid)        (Ultrogel) - Szili
kagél, "porózus üveg"               
(SelectiSphere)
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Az affinitási kromatográfia kölcsönhatásainak
tükörképi elve
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AFFINITÁSI KÖLCSÖNHATÁSOK (IMMOBILIZED LIGAND
AFFINITY TECHNIOUES)
IMMUNOAFFINITY (immunosorption) LECTINS
(glycoproteins, oligo-polysaccharides) HORMONES
(receptors/carriers/antihormones) PROTEINS
(enzymes, immunoglobulins) SUBSTRATES
(cofactors, inhibitors, modulators) NUCLEIC ACID
binding ligands (oligo(dT)cellulose) VÍRUS
binding ligands PHENYL BORONATE binding
(cis-diols) IMMOBILIZED METAL ION binding
affinity DYE-LIGAND binding affinity - Cibacron
Blue F3G-A - Malachit Green (A/T
specific) - Phenol Red (G/C specific)
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AAT
Glicin-HCl puffer, pH 2,8
perc
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LECTIN-affinitási kromatográfia
lektinek affinitási kölcsönhatásai
glikoproteinekkel - glikánokkal
N-glikozid kötésben Lectin-AAA Lectin-DSA Lectin-G
NA Lectin-MAA Lectin-SNA Lectin-PHA-L
Szerkezet specificitás L-Fuc?(l-6)GIcNAcl Galß(l-4
)GlcNAc- Man ?(l-3)62 Man- SA ?(2-3)Gal SA
?(2-6)Gal Poly-Gal-GlcNAc-
O-glikozidos kötésben Lectin-ACA Lectin-PNA Lectin
-ConA Lectin-RCA-120 Lectin-WGA
SA?(2-3)Galß(l-3)-GalNAc?-Ser/Thr Galß(l-3)
GalNAc-Ser/Thr
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AFFINITÁSI KROMATOGRÁFIA
Ready-to-use group specific adsorbents
Product Specificity Applications Eluents
Protein A-Sepharose CL-4B Fc region of IgG IgG (many species), IgG subclasses and fragments immuno complexes, antigens 1 M HAc, pH 3 0,1 M glycyltyrosine, pH 7
Con A-Sepharose ?-D-glucosyl, ?-D-mannosyl residues Glycoproteins, membrane proteins, glycolipids, polysaccharides Free sugar (methyl- ? -D-glucoside) pulse or gradient (0-0.5 M). Decreased pH (not below 3) Borate buffer (0.1 M, pH 6.5)
Lentil Lectin-Sepharose 4B Similar to Con A (weaker affinity) Membrane proteins, glycoproteins Free sugar (methyl- ? -D-glucoside) pulse or gradient (0-0.1 M)
Wheat germ Lectin-Sepharose 6MB N-acetyl-ß-D-glucos-aminyl residues Glycoproteins, polysaccharides, cells (esp. T-lymphocytes) Free sugar (N-acetyl- ß -D-glucos-amine) pulse or gradient.
Helix pomatia Lectin-Sepharose 6MB N-acetyl- ? -D galactos-aminyl residues cells (esp. T-lymphocytes), glycoproteins Free sugar (N-acetyl- ? -D-galactosamine)
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Sejtmembrán glikoprotein (Na-deoxikolát
extraktum) tisztítása L. culinaris
lectin-Sepharose 4B oszlopon
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FESTÉK LIGANDUM AFFINITÁSI KROMATOGRÁFIA DYE-LIGAN
D AFFINITY CHROMATOGRAPHY
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DYE-LIGAND AFFINITY CHROMATOGRAPHY OF HUMAN SERUM
PROTEINS
FUNCTIONAL GROUP
CIBACRON BLUE F3G-A AFFINITY PACKING
MATERIALS (Capacities ? 10 mg albumin per ml
gel) Blue Sepharose CL-6B (Pharmacia)
Affi-GelR Blue Gel           (Bio-Rad)
Fractogel TSK AF-Blue (Merck),
TOYOPEARL AF Blue HC-650M COLUMN
6x0.5cmI.D. (Pharmacia 5/5) CHROMATOGRAPHIC
SYSTEM FPLC (Pharmacia) with LCC-500 Programmer
and UV-1 Monitor (278 nm) ELUTION Equilibrated
and eluted with 10 mM phosphate buffer pH 5.8
or 6.8 Step gradient elution with 10 mM
phosphate buffer pH8.0 containing 2 M NaCl
FLOW RATE 1 ml / min SAMPLE 500 µL prepared
by Sephadex G-25 Equivalent to 50 µL of
original serum
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DYE-LIGAND AFFINITY CHROMATOGRAPHY OF HUMAN
SERUM PROTEINS
COLUMN Blue Sepharose CL-6B pH5,8 Human serum
samples prepared by Sephadex G-25
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FENIL BORONÁT AFFINITÁSI KROMATOGRÁFIA
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PSEUDO-URIDIN MEGHATÁROZÁSA HUMÁN VIZELETBEN
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PSEUDO-URIDINE DETERMINATION
PRINCIPLE NUCLEOSIDES (PSEUDO-URIDINE) WITH
VICINAL HYDROXYL GROUPS IN THEIR STRUCTURE CAN BE
EXTRACTED SELECTIVELY BY AFFINITY CHROMA-TOGRAPHY
FROM COMPLEX BIOLOGICAL SAMPLES AND SEPARATED BY
REVERSED-PHASE HPLC. SAMPLE PREPARATION MICRO-PREP
ARATIVE BORONATE AFFINITY COLUMN (Affi-Gel 601,
Bio-Rad Labs, 5x50 mm) WAS EQULIBRATED WITH 0.25M
NH4-ACETATE (pH 8-8). ONE ml URINE WAS MIXED WITH
0.3 ml 2.5M NH4-ACETATE (pH 9.5) AND CENTRIFUGED.
500 µL SUPERNATANT WAS APPLIED ON THE
AFFINITY COLUMN AND WASHED WITH 2x4 ml 0.25M NH 4
- ACETATE (pH 8.8). ELUTION WAS PERFORMED WITH 5
ml 0.1M FORMIC ACID. ELUATE WAS COLLECTED AND
FREEZE-DRYED. THE RESIDUE WAS SOLVED IN 250 µL
10mM NH4PHOSPHATE (pH 5) CONTAINING 2
METHANOL. HPLC ANALYSIS HP 1084B LIQUID
CHROMATOGRAPH ISOCRATIC ELUTION WITH 10mM
PHOSPHATE BUFFER (pH 5.1) CONTAINING 2 (v/v)
METHANOL., COLUMN ODS-HYPERSIL (25x0.46 cm, 5u)
TEMP 35C. FLOW 1 ml/min. DETECTION 254 nm.
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