Summary

Samenvatting Biochemische Technieken DEEL 1

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Course
Biochemische Technieken (AP221613)

Institution
Artesis Plantijn Hogeschool Antwerpen (Artesis)

Een zelf getypte samenvatting van deel 1 van het vak biochemische technieken. Gegeven door mevrouw Soetaert op AP hogeschool in de het derde jaar biochemie (afstudeerrichting van chemie). In de samenvatting staan bepaalde onderwerpen aangeduid die ze in de les aangaf dat mogelijke examenvragen zijn...

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Uploaded on December 17, 2024
Number of pages 48
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Type Summary

biochemie
chemie
biochemische technieken
deel1
soetaert
samenvatting
ap

Institution
Artesis Plantijn Hogeschool Antwerpen (Artesis)
Education
Biochemie
Course
Biochemische Technieken (AP221613)

LouisePeeters

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= examenvraag

Biochemische Technieken
Deel 1: Biochemische werkwijzen

Inhoudsopgave
1 Inleiding..................................................................................................... 4

2 Waterdiverse stoffen – Buffers.....................................................................5

2.1 Eigenschappen van water......................................................................................... 6
2.1.1 Inwendige structuur van water............................................................................6
2.1.2 Hydrofobe interacties..........................................................................................6
2.2 Invloed van diverse stoffen.......................................................................................6
2.2.1 Interactie met water............................................................................................6
2.2.1.1 Organische solventen....................................................................................7
2.2.1.2 Zouten........................................................................................................... 7
2.2.1.3 Ureum en guanidine-HCl................................................................................7
2.2.1.4 Chaotrope stoffen.......................................................................................... 8
2.2.2 Directe interactie met de biomolecule.................................................................8
2.2.2.1 Ionogene zepen............................................................................................. 8
2.2.2.2 Niet-ionogene zepen......................................................................................9
2.2.2.3 Zwitterionen.................................................................................................. 9
2.2.2.4 Reductantia voor disulfidebindingen...........................................................10
2.2.2.5 SH-reagentia................................................................................................ 10

3 Homogenisatie van cellen en weefsels........................................................10

3.1 Uitgangsmateriaal................................................................................................... 11
3.1.1 Soort.................................................................................................................. 11
3.1.2 Plaats................................................................................................................. 11
3.1.3 Tijdstip............................................................................................................... 11

3.2 Activiteit van hydrolytische enzymen......................................................................12
3.3 Homogenisatiemethoden........................................................................................12
3.3.1 Algemene richtlijnen.......................................................................................... 12
3.3.2 Methodes........................................................................................................... 12
3.3.2.1 Potter-Elvehjem buis....................................................................................13
3.3.2.2 Blender........................................................................................................ 13
3.3.2.3 Mortier......................................................................................................... 13
3.3.2.4 Schudden met glaskorrels...........................................................................13
3.3.2.5 Toepassing van druk...................................................................................13
3.3.2.6 Osmotische shock........................................................................................ 13
3.3.2.7 Chemisch: SDS + EDTA...............................................................................13
3.3.2.8 Ultrasonoor geluid.......................................................................................14
3.3.2.9 Enzymatisch................................................................................................ 14

,4 Zuivering van nucleïnezuren......................................................................14
4.1 Algemene strategie................................................................................................. 14
4.1.1 Uitgangsmateriaal............................................................................................. 14
4.1.2 Extractiemethoden............................................................................................ 15

4.2 Beschadiging van nucleïnezuren.............................................................................15
4.2.1 Chemische schade............................................................................................. 15
4.2.2 Biologische schade............................................................................................ 16
4.2.2.1 DNA-nucleasen of DNasen...........................................................................16
4.2.2.2 RNA-nucleasen of RNasen...........................................................................16
4.2.3 Stralingsschade................................................................................................. 17
4.2.4 Mechanische schade.......................................................................................... 17
4.3 Concentratiebepaling.............................................................................................. 17
4.3.1 UV-absorptie...................................................................................................... 17
4.3.1.1 Absorptiecentrum........................................................................................17
4.3.1.2 Extinctiecoëfficiënt bij 260 nm....................................................................17
4.3.2 Fluorescentie..................................................................................................... 18
4.3.2.1 Ethidiumbromide......................................................................................... 18
4.3.2.2 Submarine elektroforese.............................................................................18
4.3.2.3 GelRed......................................................................................................... 18
4.3.2.4 SYBRgreen................................................................................................... 19
4.2.3.5 Qubit fluorometer........................................................................................19
4.3.3 Capillaire elektroforese......................................................................................19
4.3.4 Kleurreacties...................................................................................................... 19
4.3.5 Radioactieve detectie........................................................................................19

4.4 Denaturatie en renaturatie......................................................................................20
4.4.1 Denaturatie....................................................................................................... 20
4.4.2 Renaturatie........................................................................................................ 20
4.4.3 Smelttemperatuur............................................................................................. 20

4.5 Isolatie en zuivering................................................................................................ 22
4.5.1 Manuele methodes............................................................................................ 22
4.5.1.1 Isolatie van hoogmoleculair DNA.................................................................22
4.5.1.2 Plasmide-isolatie.......................................................................................... 23
4.5.1.3 Isolatie mRNA.............................................................................................. 23
4.5.2 Kits.................................................................................................................... 24

4.6 Zuiverheid............................................................................................................... 25
4.7 Identificatie............................................................................................................. 25

4.8 Restrictie-enzymen.................................................................................................. 25
4.8.1 Endonuclease of restrictie-enzymen..................................................................25
4.8.2 Restrictie-enzymklassen....................................................................................25
4.8.3 Steractiviteit...................................................................................................... 26
4.8.4 Isochizomeren................................................................................................... 26
4.8.5 Naamgeving...................................................................................................... 26
4.8.6 Activiteit............................................................................................................ 27
4.8.7 Reactieomstandigheden....................................................................................27
4.8.8 Gevoeligheid voor dam en dcm methylatie.......................................................28
4.9 Oefeningen.............................................................................................................. 29

2
Louise Peeters Samenvatting

,5 Zuivering van eiwitten...............................................................................32
5.1 Algemene strategie................................................................................................. 32

5.2 Efficiëntie van de zuiveringsstap.............................................................................34
5.3 Concentratiebepaling (!!)........................................................................................34
5.3.1 Eenheid van concentratie..................................................................................34
5.3.2 Methoden.......................................................................................................... 35
5.3.2.1 UV-absorptie................................................................................................ 35
5.3.2.2 Biureetreactie.............................................................................................. 35
5.3.2.3 Bradford methode.......................................................................................36
5.3.2.4 Bepaling van het drooggewicht...................................................................36

5.4 Fractionering........................................................................................................... 36
5.4.1 Oplosbaarheid................................................................................................... 36
5.4.1.1 Isoëlektrische precipitatie............................................................................37
5.4.1.2 Zoutprecipitatie........................................................................................... 37
5.4.2 Ammoniumsulfaat-fractionatie..........................................................................37
5.4.2.2 Centrifugeren.............................................................................................. 38
5.4.2.3 Op punt stellen van de fractionering...........................................................39
5.4.3 Oplosmiddelfractionering...................................................................................39
5.4.4 Selectieve denaturatie.......................................................................................40
5.5 Verwijdering van zouten.......................................................................................... 40
5.5.1 Gelpermeatiechromatografie.............................................................................40
5.5.2 Filtratie.............................................................................................................. 41
5.5.2.1 Structuur..................................................................................................... 41
5.5.2.2 Karakterisering van een membraan............................................................41
5.5.2.3 Transport door een membraan....................................................................41
5.5.3 Dialyse............................................................................................................... 42

5.6 Bewaring................................................................................................................. 42
5.7 Maken van een eiwitoplossing.................................................................................43
5.7.1 Oplossen van het eiwit......................................................................................43
5.8 Detectie van denaturatie.........................................................................................43

6 Eiwitkarakterisatie....................................................................................43
6.1 Primaire structuur = AZ-sequentie..........................................................................43
6.1.1 Edman degradatie............................................................................................. 43
6.1.2 Vertaling DNA.................................................................................................... 45
6.1.3 Peptide massa ‘fingerprint’ analyse...................................................................46
6.2 Conformatie............................................................................................................. 46
6.2.1 X-stralen kristallografie......................................................................................46
6.2.2 Elektronenmicroscopie......................................................................................46
6.2.3 NMR spectroscopie............................................................................................ 46

7 Proteomics................................................................................................ 47

7.1 Wat is proteomics.................................................................................................... 47
7.2 Waarom proteomics................................................................................................ 47

7.3 Traditionele proteomics........................................................................................... 47

3
Louise Peeters Samenvatting

, 7.3.1 Tweedimensionale gelelektroforese..................................................................47
7.3.2 Beeldanalyse..................................................................................................... 47
7.3.3 Massaspectrometrie.......................................................................................... 48
7.3.3.1 Vormen van ionen.......................................................................................48
7.3.3.2 Analyse van ionen in TOF massaspectrometer............................................48
7.3.4 Bioinformatica................................................................................................... 48

7.4 Praktisch voorbeeld.................................................................................................48

1 Inleiding
 Dit schema is algemeen geldig of men eiwit isoleert uit zijn natuurlijk milieu of
aangemaakt heeft via recombinant-DNA-technologie

4
Louise Peeters Samenvatting

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