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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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Document information

  • December 17, 2024
  • December 29, 2024
  • 45
  • 2024/2025
  • Summary

Subjects

  • biochemie
  • chemie
  • biochemische technieken
  • deel1
  • soetaert
  • samenvatting
  • ap

Written for

  • Artesis Plantijn Hogeschool Antwerpen (Artesis)
  • Biochemie
  • Biochemische Technieken (AP221613)
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1  review

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By: xamdewaele • 2 weeks ago

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Good summary but the photos are staggered

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By: LouisePeeters • 2 weeks ago

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That's a shame! I'll see if I can change it:) Good luck!

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= examenvraag




Biochemische Technieken
Deel 1: Biochemische werkwijzen



Inhoudsopgave

1 Inleiding .................................................................................................................................... 5

2 Waterdiverse stoffen – Buffers.................................................................................................... 6

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 ........................................................................................................... 7
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..................................................................................................................... 10
3.1.1 Soort .................................................................................................................................. 10
3.1.2 Plaats ................................................................................................................................. 10
3.1.3 Tijdstip ................................................................................................................................ 11

3.2 Activiteit van hydrolytische enzymen .......................................................................................... 11

3.3 Homogenisatiemethoden .......................................................................................................... 12
3.3.1 Algemene richtlijnen............................................................................................................ 12
3.3.2 Methodes ........................................................................................................................... 12
3.3.2.1 Potter-Elvehjem buis ..................................................................................................... 12
3.3.2.2 Blender ........................................................................................................................ 12
3.3.2.3 Mortier ......................................................................................................................... 12
3.3.2.4 Schudden met glaskorrels ............................................................................................. 12
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 ........................................................................................................ 13

, 3.3.2.9 Enzymatisch ................................................................................................................. 13

4 Zuivering van nucleïnezuren ...................................................................................................... 14

4.1 Algemene strategie ................................................................................................................... 14
4.1.1 Uitgangsmateriaal ............................................................................................................... 14
4.1.2 Extractiemethoden .............................................................................................................. 14

4.2 Beschadiging van nucleïnezuren ................................................................................................ 15
4.2.1 Chemische schade ............................................................................................................. 15
4.2.2 Biologische schade ............................................................................................................. 15
4.2.2.1 DNA-nucleasen of DNasen ............................................................................................ 15
4.2.2.2 RNA-nucleasen of RNasen ............................................................................................ 15
4.2.3 Stralingsschade .................................................................................................................. 16
4.2.4 Mechanische schade .......................................................................................................... 16

4.3 Concentratiebepaling ............................................................................................................... 16
4.3.1 UV-absorptie....................................................................................................................... 16
4.3.1.1 Absorptiecentrum ......................................................................................................... 16
4.3.1.2 Extinctiecoëfficiënt bij 260 nm ....................................................................................... 17
4.3.2 Fluorescentie ...................................................................................................................... 17
4.3.2.1 Ethidiumbromide .......................................................................................................... 17
4.3.2.2 Submarine elektroforese ............................................................................................... 17
4.3.2.3 GelRed ......................................................................................................................... 18
4.3.2.4 SYBRgreen .................................................................................................................... 18
4.2.3.5 Qubit fluorometer ......................................................................................................... 18
4.3.3 Capillaire elektroforese ....................................................................................................... 18
4.3.4 Kleurreacties ...................................................................................................................... 18
4.3.5 Radioactieve detectie .......................................................................................................... 19

4.4 Denaturatie en renaturatie ........................................................................................................ 19
4.4.1 Denaturatie......................................................................................................................... 19
4.4.2 Renaturatie ......................................................................................................................... 19
4.4.3 Smelttemperatuur ............................................................................................................... 19

4.5 Isolatie en zuivering................................................................................................................... 21
4.5.1 Manuele methodes ............................................................................................................. 21
4.5.1.1 Isolatie van hoogmoleculair DNA ................................................................................... 21
4.5.1.2 Plasmide-isolatie .......................................................................................................... 22
4.5.1.3 Isolatie mRNA ............................................................................................................... 22
4.5.2 Kits ..................................................................................................................................... 23

4.6 Zuiverheid ................................................................................................................................ 24

4.7 Identificatie .............................................................................................................................. 24

4.8 Restrictie-enzymen ................................................................................................................... 24
4.8.1 Endonuclease of restrictie-enzymen .................................................................................... 24
4.8.2 Restrictie-enzymklassen ..................................................................................................... 24
4.8.3 Steractiviteit ....................................................................................................................... 25
4.8.4 Isochizomeren .................................................................................................................... 25
4.8.5 Naamgeving ........................................................................................................................ 25



Louise Peeters Samenvatting 2

, 4.8.6 Activiteit ............................................................................................................................. 26
4.8.7 Reactieomstandigheden...................................................................................................... 26
4.8.8 Gevoeligheid voor dam en dcm methylatie ........................................................................... 27

4.9 Oefeningen ............................................................................................................................... 28

5 Zuivering van eiwitten ............................................................................................................... 31

5.1 Algemene strategie ................................................................................................................... 31

5.2 Efficiëntie van de zuiveringsstap ................................................................................................ 32

5.3 Concentratiebepaling (!!)........................................................................................................... 32
5.3.1 Eenheid van concentratie .................................................................................................... 32
5.3.2 Methoden ........................................................................................................................... 33
5.3.2.1 UV-absorptie ................................................................................................................ 33
5.3.2.2 Biureetreactie ............................................................................................................... 33
5.3.2.3 Bradford methode ......................................................................................................... 33
5.3.2.4 Bepaling van het drooggewicht ...................................................................................... 34

5.4 Fractionering ............................................................................................................................ 34
5.4.1 Oplosbaarheid .................................................................................................................... 34
5.4.1.1 Isoëlektrische precipitatie ............................................................................................. 35
5.4.1.2 Zoutprecipitatie ............................................................................................................ 35
5.4.2 Ammoniumsulfaat-fractionatie ............................................................................................ 35
5.4.2.2 Centrifugeren ............................................................................................................... 36
5.4.2.3 Op punt stellen van de fractionering .............................................................................. 36
5.4.3 Oplosmiddelfractionering .................................................................................................... 37
5.4.4 Selectieve denaturatie ......................................................................................................... 37

5.5 Verwijdering van zouten ............................................................................................................ 38
5.5.1 Gelpermeatiechromatografie ............................................................................................... 38
5.5.2 Filtratie ............................................................................................................................... 38
5.5.2.1 Structuur ...................................................................................................................... 38
5.5.2.2 Karakterisering van een membraan ................................................................................ 38
5.5.2.3 Transport door een membraan ...................................................................................... 39
5.5.3 Dialyse ............................................................................................................................... 39

5.6 Bewaring .................................................................................................................................. 39

5.7 Maken van een eiwitoplossing ................................................................................................... 40
5.7.1 Oplossen van het eiwit ........................................................................................................ 40

5.8 Detectie van denaturatie ........................................................................................................... 40

6 Eiwitkarakterisatie .................................................................................................................... 41

6.1 Primaire structuur = AZ-sequentie.............................................................................................. 41
6.1.1 Edman degradatie ............................................................................................................... 41
6.1.2 Vertaling DNA...................................................................................................................... 42
6.1.3 Peptide massa ‘fingerprint’ analyse...................................................................................... 43

6.2 Conformatie ............................................................................................................................. 43
6.2.1 X-stralen kristallografie........................................................................................................ 43



Louise Peeters Samenvatting 3

, 6.2.2 Elektronenmicroscopie ....................................................................................................... 43
6.2.3 NMR spectroscopie ............................................................................................................. 43

7 Proteomics ............................................................................................................................... 44

7.1 Wat is proteomics ..................................................................................................................... 44

7.2 Waarom proteomics ................................................................................................................. 44

7.3 Traditionele proteomics ............................................................................................................ 44
7.3.1 Tweedimensionale gelelektroforese ..................................................................................... 44
7.3.2 Beeldanalyse ...................................................................................................................... 44
7.3.3 Massaspectrometrie ........................................................................................................... 45
7.3.3.1 Vormen van ionen ......................................................................................................... 45
7.3.3.2 Analyse van ionen in TOF massaspectrometer ............................................................... 45
7.3.4 Bioinformatica .................................................................................................................... 45

7.4 Praktisch voorbeeld .................................................................................................................. 45




Louise Peeters Samenvatting 4

,1 Inleiding
> Dit schema is algemeen geldig of men eiwit isoleert uit zijn natuurlijk milieu of aangemaakt heeft
via recombinant-DNA-technologie
> De gekozen zuiveringsmethoden zijn afhankelijk van de grootte en aard van de moleculen




Louise Peeters Samenvatting 5

, 2 Waterdiverse stoffen – Buffers

2.1 Eigenschappen van water
> Water is een molecule met een sterk dipoolmoment en vormt makkelijk waterstofbruggen, dit
resulteert in volgende eigenschappen:
» Grote diëlectriciteitsconstante (permeativiteit)
» Goed oplosmiddel voor polaire stoffen
» Hoge verdampingswarmte
» Hoge oppervlaktespanning
» Bijzondere inwendige structuur!!
» Optreden van hydrofobe interacties!!

2.1.1 Inwendige structuur van water
> Groot verschil in ENW tussen O en H à sterk dipoolmoment
> Vrije elektronenparen op O vormen samen met H de hoekpunten van een tetraëder
> In water vormen zich clusters van watermoleculen met een sterke inwendige structuratie:
entropie (S) neemt af

2.1.2 Hydrofobe interacties
> Vaststellingen:
» Apolaire ketens gaan associëren bij oplossing in H2O
» Eiwitten + nucleïnezuren komen in bepaalde conformatie voor in water
» Eiwit/peptide neemt na synthese zeer geordende biologisch actieve conformatie aan
> Verklaring:
» Dit is thermodynamisch!
» Men streeft naar een minimale inwendige energie (G) en zo groot mogelijk entropie (S)
» Als keten niet oprolt: sterke structuratie rond hydrofobe zijketens, dit is entropisch niet
gunstig
» Als keten wel oprolt: wordt compacter, de bewegingsvrijheid stijgt en dit is entropisch
gunstig
» Waterstofbrugvorming van H2O met aminozuurzijketens op uitwendige oppervlak van
eiwit versterken conformatie
> De stabiliteit vd conformatie is door het entropie-effect (dat hydrofobe groepen naar elkaar drijft)
en de VDW-krachten tussen apolaire groepen of de extra H-bruggen tussen watermoleculen
> Is een zeer labiel evenwicht, biomoleculen kan makkelijk denatureren

2.2 Invloed van diverse stoffen
> In water neigt biomolecule zich te ontrollen en streeft H2O naar een maximumaantal
waterstofbruggen
> Een stof kan de conformatie van een biomolecule beïnvloeden door:
» Interactie met water: wijziging structuur/conformatie van biomolecule
» Directe interactie met biomolecule

2.2.1 Interactie met water
> Organische stoffen hebben een verzwakkende en zouten een versterkende werking op:
» Onderlinge aantrekking tussen watermoleculen
» Oppervlaktespanning



Louise Peeters Samenvatting 6

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