Samenvatting Labtools
,Inhoud
1. Basis begrippen voor scheidingsmethoden, gelfiltratie en affiniteitschromatografie .................. 5
1.1 Kolom chromatografie .................................................................................................................. 6
1.2 Gelfiltratie ..................................................................................................................................... 8
1.3 Affiniteit ........................................................................................................................................ 9
1.3.1 Het taggen van een eiwit ..................................................................................................... 10
1.3.2 Intermezzo antilichamen ..................................................................................................... 11
1.3.3 Streptavidine-biotine ........................................................................................................... 13
1.3.4 Samenvatting voorbeelden affiniteitschromatografie ........................................................ 13
2. Scheiding op basis van polariteit en elektroforese ................................................................... 15
2.1 Papier dunne laag chromatografie ............................................................................................. 15
2.2 Adsorptie chromatografie en Partitie chromatografie. .............................................................. 15
2.2.1 Adsorptie chromatografie .................................................................................................... 15
2.2.2 Partitie chromatografie ........................................................................................................ 16
2.3 Elektroforese theorie .................................................................................................................. 17
2.3.1 Mobiliteit (µ) ........................................................................................................................ 18
2.3.2 Migratiesnelheid (v) ............................................................................................................. 18
2.4 Typen elektroforese .................................................................................................................... 19
2.4.1 DNA elektroforese................................................................................................................ 19
2.4.2 Eiwit elektroforese: Eigen lading (iso-elektrisch punt, natief) ............................................. 19
2.4.3 Eiwit elektroforese: Toegevoegde negatieve lading (SDS-PAGE, denaturerend) ................ 21
2.4.4 Eiwit elektroforese: Combinatie IEF en SDS-PAGE (2D elektroforese) ................................ 23
3. Ionenwisselingschromatografie .............................................................................................. 24
3.1 Anion of kationwisseling ............................................................................................................. 24
3.2 Berekenen van lading van aminozuren/peptiden/eiwitten........................................................ 26
3.3 Toepassing scheiden op basis van lading .................................................................................... 28
4. Lichtmicroscopie .................................................................................................................... 30
4.1 Lenzen ......................................................................................................................................... 30
4.2 Het oog ........................................................................................................................................ 32
4.3 Lenzen in microscopen ............................................................................................................... 34
4.3.1 Stralengang .......................................................................................................................... 34
4.3.2 Scheidend vermogen (resolutie) .......................................................................................... 34
4.3.4 Optische beeldfouten .......................................................................................................... 36
4.4 Beperkingen lichtmicroscoop en donker veld microscopie ........................................................ 37
4.4.1 Beperkingen lichtmicroscoop .............................................................................................. 37
2
, 4.4.2 Donkerveld microscopie ...................................................................................................... 38
5. Overige microscoop technieken; fluorescentie deel l ............................................................... 39
5.1 fasecontrast ................................................................................................................................ 39
5.1.1 Versterking en uitdoving van golven ................................................................................... 39
5.2 Elektronenmicroscopie ............................................................................................................... 41
5.2.1 Transmissie-elektronenmicroscopie (TEM) ......................................................................... 41
5.2.2. Scanning elektronenmicroscopie (SEM) ............................................................................. 42
5.3 Fluorescentie microscopie deel 1 ............................................................................................... 43
5.3.1 Principe van labeling met fluoroforen ................................................................................. 44
5.3.2 Fluorescentie meten ............................................................................................................ 45
6. Fluorescentie deel ll; flow cytometrie ..................................................................................... 46
6.1 Confocal microscopie .................................................................................................................. 46
6.2 FRAP, FLIP en FRET ...................................................................................................................... 47
6.2.1 FRAP (Fluorescence recovery after photobleaching)........................................................... 47
6.2.2 FLIP (Fluorescence loss in photobleaching) ......................................................................... 47
6.2.3 FRET (Förster resonance energy transfer) ........................................................................... 47
6.3 Flow cytometrie .......................................................................................................................... 49
6.3.1 Fluidics systeem ................................................................................................................... 49
6.3.2 Optics system ....................................................................................................................... 49
6.3.3 Electronics system................................................................................................................ 49
6.3.4 Grootte en complexiteit van deeltjes meten ....................................................................... 50
6.3.5 Intensiteit van fluorescentie meten..................................................................................... 52
6.3.6 FACS (Fluorescence Activated Cell Sorting) ......................................................................... 53
7. Centrifugeren ......................................................................................................................... 54
7.1 Bezinksnelheid en wrijving .......................................................................................................... 54
7.2 Centripetaal kracht ..................................................................................................................... 55
7.3 Bouw centrifuge en tareren ........................................................................................................ 56
7.4 Centrifugetechnieken ................................................................................................................. 57
7.5 Typen centrifuges en rotors ........................................................................................................ 58
8. Massaspectrometrie............................................................................................................... 59
8.1 Principe massaspectrometrie ..................................................................................................... 59
8.1.1 Atoommassa ........................................................................................................................ 61
8.1.2 m/z waarde .......................................................................................................................... 62
8.1.3 Grafiek aflezen en definities ................................................................................................ 62
8.1.4 Isotopen ............................................................................................................................... 63
8.2 Ionisatietechnieken ..................................................................................................................... 65
3
, 8.3 Bouw van de MS en typen MS .................................................................................................... 67
8.3.1 Quadropole massa analyser................................................................................................. 68
8.3.2 Ion trap mass analyser ......................................................................................................... 68
8.3.3 Time-of-flight (TOF) mass analyser ...................................................................................... 68
8.3 Tandem-MS (MS/MS) ................................................................................................................. 69
8.5 Voorscheiding (GC-MS, LC-MS) ................................................................................................... 69
4
,Inhoud
1. Basis begrippen voor scheidingsmethoden, gelfiltratie en affiniteitschromatografie .................. 5
1.1 Kolom chromatografie .................................................................................................................. 6
1.2 Gelfiltratie ..................................................................................................................................... 8
1.3 Affiniteit ........................................................................................................................................ 9
1.3.1 Het taggen van een eiwit ..................................................................................................... 10
1.3.2 Intermezzo antilichamen ..................................................................................................... 11
1.3.3 Streptavidine-biotine ........................................................................................................... 13
1.3.4 Samenvatting voorbeelden affiniteitschromatografie ........................................................ 13
2. Scheiding op basis van polariteit en elektroforese ................................................................... 15
2.1 Papier dunne laag chromatografie ............................................................................................. 15
2.2 Adsorptie chromatografie en Partitie chromatografie. .............................................................. 15
2.2.1 Adsorptie chromatografie .................................................................................................... 15
2.2.2 Partitie chromatografie ........................................................................................................ 16
2.3 Elektroforese theorie .................................................................................................................. 17
2.3.1 Mobiliteit (µ) ........................................................................................................................ 18
2.3.2 Migratiesnelheid (v) ............................................................................................................. 18
2.4 Typen elektroforese .................................................................................................................... 19
2.4.1 DNA elektroforese................................................................................................................ 19
2.4.2 Eiwit elektroforese: Eigen lading (iso-elektrisch punt, natief) ............................................. 19
2.4.3 Eiwit elektroforese: Toegevoegde negatieve lading (SDS-PAGE, denaturerend) ................ 21
2.4.4 Eiwit elektroforese: Combinatie IEF en SDS-PAGE (2D elektroforese) ................................ 23
3. Ionenwisselingschromatografie .............................................................................................. 24
3.1 Anion of kationwisseling ............................................................................................................. 24
3.2 Berekenen van lading van aminozuren/peptiden/eiwitten........................................................ 26
3.3 Toepassing scheiden op basis van lading .................................................................................... 28
4. Lichtmicroscopie .................................................................................................................... 30
4.1 Lenzen ......................................................................................................................................... 30
4.2 Het oog ........................................................................................................................................ 32
4.3 Lenzen in microscopen ............................................................................................................... 34
4.3.1 Stralengang .......................................................................................................................... 34
4.3.2 Scheidend vermogen (resolutie) .......................................................................................... 34
4.3.4 Optische beeldfouten .......................................................................................................... 36
4.4 Beperkingen lichtmicroscoop en donker veld microscopie ........................................................ 37
4.4.1 Beperkingen lichtmicroscoop .............................................................................................. 37
2
, 4.4.2 Donkerveld microscopie ...................................................................................................... 38
5. Overige microscoop technieken; fluorescentie deel l ............................................................... 39
5.1 fasecontrast ................................................................................................................................ 39
5.1.1 Versterking en uitdoving van golven ................................................................................... 39
5.2 Elektronenmicroscopie ............................................................................................................... 41
5.2.1 Transmissie-elektronenmicroscopie (TEM) ......................................................................... 41
5.2.2. Scanning elektronenmicroscopie (SEM) ............................................................................. 42
5.3 Fluorescentie microscopie deel 1 ............................................................................................... 43
5.3.1 Principe van labeling met fluoroforen ................................................................................. 44
5.3.2 Fluorescentie meten ............................................................................................................ 45
6. Fluorescentie deel ll; flow cytometrie ..................................................................................... 46
6.1 Confocal microscopie .................................................................................................................. 46
6.2 FRAP, FLIP en FRET ...................................................................................................................... 47
6.2.1 FRAP (Fluorescence recovery after photobleaching)........................................................... 47
6.2.2 FLIP (Fluorescence loss in photobleaching) ......................................................................... 47
6.2.3 FRET (Förster resonance energy transfer) ........................................................................... 47
6.3 Flow cytometrie .......................................................................................................................... 49
6.3.1 Fluidics systeem ................................................................................................................... 49
6.3.2 Optics system ....................................................................................................................... 49
6.3.3 Electronics system................................................................................................................ 49
6.3.4 Grootte en complexiteit van deeltjes meten ....................................................................... 50
6.3.5 Intensiteit van fluorescentie meten..................................................................................... 52
6.3.6 FACS (Fluorescence Activated Cell Sorting) ......................................................................... 53
7. Centrifugeren ......................................................................................................................... 54
7.1 Bezinksnelheid en wrijving .......................................................................................................... 54
7.2 Centripetaal kracht ..................................................................................................................... 55
7.3 Bouw centrifuge en tareren ........................................................................................................ 56
7.4 Centrifugetechnieken ................................................................................................................. 57
7.5 Typen centrifuges en rotors ........................................................................................................ 58
8. Massaspectrometrie............................................................................................................... 59
8.1 Principe massaspectrometrie ..................................................................................................... 59
8.1.1 Atoommassa ........................................................................................................................ 61
8.1.2 m/z waarde .......................................................................................................................... 62
8.1.3 Grafiek aflezen en definities ................................................................................................ 62
8.1.4 Isotopen ............................................................................................................................... 63
8.2 Ionisatietechnieken ..................................................................................................................... 65
3
, 8.3 Bouw van de MS en typen MS .................................................................................................... 67
8.3.1 Quadropole massa analyser................................................................................................. 68
8.3.2 Ion trap mass analyser ......................................................................................................... 68
8.3.3 Time-of-flight (TOF) mass analyser ...................................................................................... 68
8.3 Tandem-MS (MS/MS) ................................................................................................................. 69
8.5 Voorscheiding (GC-MS, LC-MS) ................................................................................................... 69
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