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Samenvatting Geïntegreerd practicum 1 (biochemie): practicumverslag
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Verslag van alle uitgevoerde experimenten tijdens de practica voor het vak geïntegreerd practicum: biochemie uit de 2e bachelor.
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GEÏNTEGREERD PRACTICUMBIOCHEMIE: VERSLAGEN
EXPERIMENTEN 1-5
Academiejaar 2019-2020
Bronnen:
Resultaten afkomstig van eigen bevindingen tijdens het practicum. Theoretische uitleg gebaseerd
op de uitleg en PowerPoint gegeven tijdens de inleidingsles door Prof. Eva Geuens.
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,Inhoudsopgave
Experiment 1: bepaling succinaat dehydrogenase (SDH) ............................................................................. 5
1. Inleiding en doel .................................................................................................................................... 5
2. Materiaal en methode........................................................................................................................... 5
2.1 Oplossingen ..................................................................................................................................... 5
2.2 Verdunningsreeks TTC ..................................................................................................................... 6
3. Resultaten.............................................................................................................................................. 6
3.1 TTC-ijklijn ......................................................................................................................................... 6
3.2 SDH-assay: bepaling van SDH-activiteit in de subcellulaire fracties................................................ 7
3.2.1 Gemeten OD-waarden ............................................................................................................. 7
3.2.2 Gereduceerde TTC .................................................................................................................... 7
3.2.3 Drooggewicht ........................................................................................................................... 8
3.2.4 Berekening van de uiteindelijke SDH-activiteit ........................................................................ 9
4. Conclusie ............................................................................................................................................... 9
4.1 Histogram ........................................................................................................................................ 9
Experiment 2: afzonderen van het LDH-5 iso-enzym uit leverweefsel ....................................................... 10
1. Inleiding en doel .................................................................................................................................. 10
2. Materiaal en methode......................................................................................................................... 11
2.1 Oplossingen ................................................................................................................................... 11
2.2 Verdunningsreeks BSA................................................................................................................... 14
3. Resultaten............................................................................................................................................ 15
3.1 Ammoniumsulfaat precipitatie van de cytosolfractie ................................................................... 15
3.2 Dialyse ........................................................................................................................................... 15
3.3 Testen van de eiwit en enzym concentratie van de verschillende fracties ................................... 16
3.3.1 LDH-enzym assay van de weefselfracties ............................................................................... 16
3.3.1.1 Fracties: gemeten absorptie............................................................................................ 16
3.3.1.2 Fracties: absorptie per minuut ........................................................................................ 16
3.3.1.3 Fracties: LDH-concentratie (units per liter) ..................................................................... 17
3.3.2 Eiwittest van de weefselfacties: methode van Bradford ....................................................... 17
3.3.2.1 BSA-ijklijn voor LDH ......................................................................................................... 17
3.3.2.2 Fracties: gemeten OD-waarden ...................................................................................... 18
3.3.2.3 Fracties: eiwitconcentratie .............................................................................................. 18
3.3.3 AM-pool .................................................................................................................................. 19
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, 3.4 Ionenuitwisselingschromatografie ................................................................................................ 19
3.5 Testen van de eiwit en enzym concentratie van de opgevangen stalen ...................................... 19
3.5.1 LDH-enzym assay van de opgevangen stalen ......................................................................... 20
3.5.1.1 Opgevangen stalen: gemeten absorptie ......................................................................... 20
3.5.1.2 Opgevangen stalen: absorptie per minuut...................................................................... 20
3.5.1.3 Opgevangen stalen: LDH-concentratie (units per liter) .................................................. 21
3.5.2 Eiwittest van de opgevangen stalen: methode van Bradford ................................................ 21
3.5.2.1 Opgevangen stalen: gemeten OD-waarden .................................................................... 21
3.5.2.2 Opgevangen stalen: eiwitconcentratie ........................................................................... 22
3.6 SDS-PAGE ....................................................................................................................................... 22
3.6.1 SDS-PAGE gel 1 ....................................................................................................................... 22
3.6.1.1 Staalvoorbereiding gel 1 .................................................................................................. 22
3.6.1.2 Resultaat SDS-PAGE gel 1 ................................................................................................ 23
3.6.2 SDS-PAGE gel 2 ....................................................................................................................... 23
3.6.2.1 Staalvoorbereiding gel 2 .................................................................................................. 23
3.6.2.2 Resultaat SDS-PAGE gel 2 ................................................................................................ 24
3.6.2.3 Opstellen van de merker-ijklijn ....................................................................................... 25
3.6.2.4 Bepaling moleculair gewicht van de LDH-eiwitten ......................................................... 25
3.7 LDH iso-enzym scheiding ............................................................................................................... 26
3.8 Western blotting............................................................................................................................ 27
4. Conclusie ............................................................................................................................................. 28
4.1 Testen van de eiwit en enzym concentratie van de verschillende fracties ................................... 28
4.2 Testen van de eiwit en enzym concentratie van de opgevangen stalen ...................................... 28
4.3 SDS-PAGE ....................................................................................................................................... 28
4.3.1 Gel 1........................................................................................................................................ 28
4.3.2 Gel 2........................................................................................................................................ 28
4.4 LDH iso-enzym scheiding ............................................................................................................... 29
4.5 Western Blotting ........................................................................................................................... 29
Experiment 3: electroforetische scheidingen van serum op SDS-PAGE...................................................... 30
1. Inleiding en doel .................................................................................................................................. 30
2. Materiaal en methode......................................................................................................................... 30
2.1 Oplossingen (idem oplossingen SDS-PAGE experiment 2) ............................................................ 30
3. Resultaten............................................................................................................................................ 32
2
, 3.1 Staalvoorbereiding ........................................................................................................................ 32
3.2 SDS-PAGE ....................................................................................................................................... 32
3.2 Bepaling van de onbekende eiwitten ............................................................................................ 33
4. Conclusie ............................................................................................................................................. 33
Experiment 4: fluorimetrische bepaling van kininesulfaat ......................................................................... 34
1. Inleiding en doel .................................................................................................................................. 34
2. Materiaal en methode......................................................................................................................... 34
2.1 Registreren van een uitdovingscurve van kinine sulfaat: verdunningsreeks kaliumjodide .......... 34
2.2 Opstellen van een kininesulfaat calibratiecurve in af- en aanwezigheid van jodide .................... 35
2.2.1 Verdunningsreeks kininesulfaat ............................................................................................. 35
2.2.2 Twee nieuwe reeksen aanmaken ........................................................................................... 35
2.3 Multiple standaard-additiemethode: concentratiebepaling toegevoegd kininesulfaat ............... 35
3. Resultaten............................................................................................................................................ 36
3.1 Registreren van een dovingscurve van kininesulfaat .................................................................... 36
3.2 Opstellen van een kininesulfaat calibratiecurve in af- en aanwezigheid van jodide .................... 36
3.3 Multiple standaard-additiemethode ............................................................................................. 37
3.3.1 Onbekende staal: berekening van de initiële kininesulfaatconcentratie ............................... 38
4. Conclusie ............................................................................................................................................. 38
4.1 Dovingscurve ................................................................................................................................. 38
4.2 Kalibratiecurve............................................................................................................................... 38
4.3 Multiple standaardadditiemethode .............................................................................................. 38
Experiment 5: bepalen van de Km en Vm van lactaat dehydrogenase via een kinetische methode ......... 39
1. Inleiding en doel .................................................................................................................................. 39
2. Materiaal en methode......................................................................................................................... 39
2.1 Oplossingen ................................................................................................................................... 39
2.2 Verdunningsreeksen ...................................................................................................................... 40
2.2.1 Verdunningsreeks van het substraat...................................................................................... 40
2.2.2 Verdunningsreeks van het enzym .......................................................................................... 40
3. Resultaten............................................................................................................................................ 41
3.1 Bepalen van het pH-optimum van lactaat dehydrogenase ........................................................... 41
3.1.1 Absorptieveranderingen over de tijd bij verschillende pH-buffers ........................................ 41
3.1.2 Bepaling pH-optimum ............................................................................................................ 41
3.2 Enzymactiviteit in functie van substraatconcentratie ................................................................... 42
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