2. MATERIALS AND METHODS
2.1 M ATERIALS
2.1.1 Chemicals, kits and buffers
Standard chemicals used in this study were obtained from AppliChem (Darmstadt, Germany), BD (Heidelberg, Germany), Roche (Mannheim, Germany), Merck, Novagen and Calbiochem (Darmstadt, Germany), Roth (Karlsruhe, Germany), Serva (Heidelberg, Germany) and Sigma-Aldrich (Taufkirchen, Germany). The complete list of individual reagents can be found in Table 5.
Table 5 List of chemicals used in this study and their suppliers.
Chemical Supplier
[35S]-L-methionine Hartmann Analytic
2-mercaptoethanol Sigma-Aldrich
6-aminocaproic acid Sigma-Aldrich
Acetic acid Roth
Acetone Roth
Acrylamide/bisacrylamide (37.5:1) solution Roth Acrylamide, 4x crystallised Roth
Agarose NEEO ultra-quality Roth
Ammonium persulfate Roth
Ampicillin AppliChem
Antimycin Sigma-Aldrich
ATP Roche
Bacto Agar BD
Bacto Peptone BD
BactoTryptone BD
Bacto Yeast Extract BD
Bio-Rad Protein Assay Bio-Rad
Bis-Acrylamide Roth
Bis-Tris Buffer grade AppliChem
Bovine serum albumin Sigma-Aldrich
Bromophenol Blue Merck
BS2G Thermo Scientific
BS3 Thermo Scientific
Complete EDTA-free protease inhibitor mix Roche Coomassie Brilliant Blue G-250 Roth Coomassie Brilliant Blue R-250 Roth
Creatine kinase Roche
Creatine phosphate Roche
CSM-amino acid MP Biomedicals
D-Desthiobiotin IBA
DMSO AppliChem
DNA ladder mix “Gene Ruler” Thermo Scientific
DSG Thermo Scientific
DSS Thermo Scientific
DTT Roth
EDTA Roth
Ethanol Roth
Ethidium bromide 0.025% w/v Roth
Galactose, D(+) Roth
Glucose, D(+) Roth
GDN101 Anatrace
L-Glutathione reduced Sigma Aldrich
Glutaraldehyde EM Grade Polysciences
Glycerol Sigma-Aldrich
Glycine Roth
HEPES Roth
Herring sperm DNA Promega
Hydrochloric acid 37% w/v Roth
Materials and Methods
Imidazole Sigma Aldrich
IPTG Sigma Aldrich
KanamycinSulfate Sigma Aldrich
Lithium acetate AppliChem
LMNG Anatrace
Magnesium chloride heptahydrate Merck
Methanol Roth
Methionine Roth
MitoTracker Orange CMTMRos Thermo Scientific
MOPS Sigma-Aldrich
NADH Roche
Ni2+-NTA agarose Macherey-Nagel
Oligomycin Sigma-Aldrich
Ortho-phosphoric acid Merck
PEG-4000 (polyethylene glycol 4000) Merck
PMSF Roth
Potassium chloride Roth
Potassium dihydrogen phosphate Roth Potassium hydrogen diphosphate Roth
Proteinase K Roche
Restriction Enzymes Thermo Scientific
Roti-Quant Roth
SDS Roth
SDS-PAGE protein standard Serva
Lipodisq SMA Copolymer 3:1 Sigma-Aldrich
Sodium chloride Roth
Sodium hydroxide AppliChem
Sorbitol Roth
Strep-tactin Sepharose 50% suspension IBA
Sucrose Roth
TCA Merck
TEMED Roth
Tricine Roth
Tris Roth
Tween-20 Roth
Urea Roth
Valinomycin Sigma-Aldrich
Yeast nitrogen base without amino acids BD
Zymolase 20 T Seikagaku Biobusiness Corporation
2.1.1.2 Kits
Commercial kits used in this study are listed in Table 6. They were used according to the instructions from the manufacturer.
Table 6 List of commercial kits used in this study along with their manufacturer.
Kit Supplier
Alkaline phosphatase, shrimp Roche Applied Science ECL Plus Western Blotting Detection
Reagent
Thermo Scientific FastDigest restriction enzymes Thermo Scientific Flexi Rabbit Reticulocyte Lysate
System Promega
Immobilon-P Transfer membrane Millipore KOD Hot Start DNA Polymerase Merck mMessagemMachine SP6
transcription Kit
Invitrogen
Rapid DNA Ligation Kit Thermo Scientific TNT Quick Coupled Transcription/
Translation SP6
Promega Wizard SV Gel and PCR Clean-Up Promega
Wizard SV Mini-Prep Promega
2.1.1.3 Buffer and Media Recipes
Recipes of the buffers and culture media used is provided in Table 7. All solutions were prepared using analytical grade chemicals. Media for bacteria and yeast growth was autoclaved post preparation.
Table 7 List of commonly used buffers in this study along with their composition.
Buffer Components
Amino acid-free media 0.67% (w/v) yeast nitrogen base w/o amino acids, 2%
Materials and Methods
sugar, 0.07% CSM-amino acid Ampicillin 100 mg/ml, sterile filtered
AVO mix 1 mM antimycin, 0.1 mM valinomycin, 2 mM
oligomycin in ethanol Bacteria Lysis Buffer
40 mM Tris/HCl, pH 7.4, 500 mM NaCl, 10 mM Imidazole, 1 mg/ml lysozyme, 0.5 mM PMSF and Complete EDTA-free protease inhibitor mix BN anode buffer 50 mM Bis-Tris pH 7.0
BN cathode buffer 50 mM tricine, 15 mM Bis-Tris, with or without 0.02%
Coomassie Brilliant Blue G-250
BN gel buffer 67 mM 6-aminocaproic acid, 50 mM Bis-Tris pH 7.0 BN sample loading buffer 0.5% (w/v) Coomassie Brilliant Blue G-250, 50 mM
6-aminocaproic acid, 10 mM Bis-Tris pH 7.0 Carrier DNA Herring sperm DNA (10 mg/ml) in TE buffer Colloidal Coomassie staining
solution
0.1% (w/v) Coomassie Brilliant Blue G-250, 2% (w/v) phosphoric acid, 10% (w/v) ammonium sulfate, 20%
methanol
Coomassie staining solution 1.5 g/l Coomassie Brilliant Blue R-250, 40% ethanol, 10% acetic acid
Destaining solution 30% ethanol, 10% acetic acid
Detergent solubilisation buffer 20 mM HEPES/KOH pH 7.4, 150 mM NaCl, 10%
glycerol, 0.1 mM EDTA, 1% detergent
DTT buffer 10 mM DTT, 100 mM Tris/HCl pH 9.4
EDTA 0.5 M EDTA, pH 8.0
Glutaraldehyde 25% solution, EM grade
10% Glycerol buffer 10% glycerol, 20 mM HEPES/KOH, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, detergent as indicated
30% Glycerol buffer
30% glycerol, 20 mM HEPES/KOH, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, detergent as indicated,
glutaraldehyde as indicated
Homogenisation buffer 0.6 M sorbitol, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 0.2% (w/v) fatty acid free BSA, 1 mM PMSF
Import buffer
3% (w/v) fatty acid free bovine serum albumin, 250 mM sucrose, 80 mM KCl, 5 mM MgCl2, 10 mM MOPS-KOH pH 7.2, 5 mM methionine, 2 mM KH2PO4, 2 mM ATP, 2 mM NADH, with or without 100 μg/ml creatine kinase (CK) and 5 mM creatine phosphate (CP) LB medium 1% (w/v) tryptone, 0.5% (w/v) NaCl, 1% (w/v) yeast
extract
LB Agar LB medium with 1.5% (v/w) agar, sterilised by autoclaving (20 min, 120°C)
Lithium acetate/PEG 0.1 M lithium acetate, 40% polyethylene glycol 4000 in water, filter sterilised
Methotrexate 10 mM stock in water, used as 2000X, stored frozen at -20°C in single-use aliquots
PMSF stock 0.2 M PMSF in ethanol
Potassium phosphate buffer
pH 7.4 80.2% (w/v) K2HPO4, 19.8% (w/v) KH2PO4
Resolving gel (for SDS-PAGE) 10-16% acrylamide, 80 mM Tris-HCl pH 6.8, 0.1%
SDS, 0.1% APS, 0.05% TEMED
SDS Protein loading dye (PLD) 10% glycerol, 2% (w/v) SDS, 0.01% bromophenol blue, 60 mM Tris/HCl pH 6.8
SDS running buffer 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS
SEM 250 mM sucrose, 10 mM MOPS-KOH pH 7.2, 1 mM
EDTA
SMA solubilisation buffer 50 mM HEPES/KOH pH 7.4, 150 mM NaCl, 5 mM Imidazole, 0.2 mM PMSF, 20 mg SMA/ml of buffer SMA wash buffer 50 mM HEPES/KOH pH 7.4, 150 mM NaCl, 5 mM
Imidazole
Stacking gel (for SDS-PAGE) 4% (w/v) arylamide, 0.1% (w/v) SDS, 380 mM Tris-HCl pH 8.8, 0.1% (w/v) APS, 0.05% TEMED TAE buffer 40 mM Tris/acetate pH 8.0, 2 mM EDTA
TBST 50 mM Tris, 150 mM NaCl, 0.05% Tween-20
TBS 50 mM Tris, 150 mM NaCl
TCA solution 72% trichloracetic acid in water
Transfer Buffer 25 mM Tris, 192 mM glycine, 10% ethanol Wash buffer 20 mM HEPES/KOH pH 7.4, 150 mM NaCl, 10%
glycerol, 1 mM PMSF, 0.1 mM EDTA, detergent as indicated
YPAD (2x) 2% (w/v) yeast extract, 4% (w/v) peptone, 4% (w/v) glucose, 0.008% (w/v) adenine hemisulfate,
autoclaved
YPD 1% yeast extract, 2% peptone, 2% glucose,
autoclaved
YPG 1% (w/v) yeast extract, 2% (w/v) peptone, 3% (w/v) glycerol, autoclaved
Zymolyase buffer 1.2 M sorbitol, 20 mM potassium phosphate pH 7.4