· RNeasy Micro Kit (Qiagen)
· DEPC-treated water (Invitrogen)
· IGEPAL® CA-630 for molecular biology (Sigma) CAUTION May develop turbidity or sediment during storage. A clear liquid can be obtained on heating to 40 ° C.
· Random Primers (Invitrogen)
· dNTP Set, 100 mM each A,C,G,T (GE Healthcare)
· RNasin® Plus RNase Inhibitor (Promega)
· Prime RNase Inhibitor (Eppendorf)
· SuperScript® III Reverse Transcriptase (Invitrogen)
· Taq DNA Polymerase (Roche)
· Agarose (Biozym)
· Trizma® base (Sigma) CAUTION It is irritating to eyes.
· Boric acid (Sigma) CAUTION It is toxic.
· EDTA, Titriplex® III (Merck)
· Ethidiumbromide, 1% stock solution (Carl Roth) CAUTION It has mutagenic properties and is irritating.
· Glycerol, ROTIPURAN® (Carl Roth)
· Bromophenol Blue (Serva)
· Xylene Cyanole FF (Bio-Rad) CAUTION It is irritating to eyes, respiratory system and skin.
· 100 bp DNA ladder (New England Biolabs)
· EasyPure® DNA Purification Kit (Biozym)
· TOPO-TA Cloning® Kit (Invitrogen)
· BactoTM Trypton (Becton Dickinson)
· BactoTM Yeast Extract (Becton Dickinson)
· BactoTM Agar (Becton Dickinson)
· Ampicillin sodium salt (Sigma)
· 5-Bromo-4-chloro-3-indoyl ß-D-galactopyranoside (X-Gal, Sigma)
· DirectPrep 96 Miniprep Kit (Qiagen)
· NaH2PO4 x H2O (Carl Roth)
· Na2HPO4 (Carl Roth)
· NaCl (Merck)
· Dynabeads® Protein G (Invitrogen)
· Na-acetate (Merck)
· SDS (Carl Roth) CAUTION It is harmful. Avoid inhalation and skin contact.
· MEGA-10 (Merck)
· N-Glycosidase F recombinant (Roche)
· Urea (Sigma)
· Thiourea (Sigma) CAUTION It is harmful if swallowed.
· PharmalyteTM, broad range pH 3-10 (GE Healthcare)
· ImmobilineTM Dry Strip, pH 3-10, 24 cm (GE Healthcare)
· SDS-PAGE Molecular Weight Standard, broad range (Bio-Rad)
· Brilliant Blue R 250 (Sigma)
· Methanol (Sigma) CAUTION It is flammable.
· Acetic acid (Sigma) CAUTION It is corrosive and irritating.
· Ammonium bicarbonate (NH4HCO3, Sigma)
· DL-Dithiothreitol (DTT, Sigma) CAUTION It is toxic and may cause irritation to skin and respiratory system.
· Acetonitrile (Sigma) CAUTION It is flammable and harmful.
· Jodoacetamide (Sigma) CAUTION It is toxic.
· Trypsin modified from porcine pancreas (Serva)
· Trifluoroacetic acid (TFA, Applied Biosystems) CAUTION It is harmful and corrosive.
· alpha-Cyano-4-Hydroxycinnamic acid (Bruker Daltonics) CAUTION It is harmful.
Reagent Setup
· TBE (10x): 0.89 M Tris, 0.89 M Boric acid, 0.02 M EDTA, pH 8.0; Store at room temperature.
· DNA sample buffer (6x): 50% (v/v) Glycerol, 0.02% (w/v) Bromphenol blue, 0.02% (w/v) Xylencyanol FF, 10 mM Tris, pH 7.5; Store at 4 ° C.
· LB-Medium: 1% (w/v) BactoTM Trypton, 0.5% (w/v), BactoTM Yeast Extract, 0.5% (w/v) NaCl; Autoclave and store at room temperature.
· LB-Agar: LB-Medium with 1.5% (w/v) BactoTM Agar, supplied with 100 μg/ml Ampicillin; Autoclave and store plates at 4 ° C.
· PBS (10x): 18.8 mM NaH2PO4 x H2O, 83.8 mM Na2HPO4, 1,5 M NaCl; Store at room temperature. Check pH after dilution to 1x.
· Washing buffer for Dynabeads® Protein G: 0.1 M Na-acetate, 0.15 M NaCl, pH 5.0; store at room temperature. Check pH before use.
· Elution buffer for Dynabeads® Protein G: 1% (w/v) SDS in PBS; Store at room temperature.
· Deglycosylation buffer: PBS with 1% (w/v) MEGA-10, pH 7.2; Store at room temperature.
CRITICAL In case of MEGA-10 precipitates heat buffer while stirring until detergent has dissolved completely. Let cool down to room temperature and check pH before use.
· Rehydration buffer for IPG strips: 8 M Urea, 2 M Thiourea, 2% (v/v) PharmalyteTM 3-10, Bromphenol blue; Prepare a fresh solution for each experiment.
· IEF sample buffer: 6 M Urea, 2 M Thiourea, 10% (v/v) Glycerol, Bromphenol blue; Prepare a fresh solution for each experiment.
· Equilibration buffer for 2D gel: 6 M Urea, 4% (w/v) SDS, 30% (w/v) Glycerol, 50 mM Tris, Bromphenol blue; Prepare a fresh solution for each experiment.
· Coomassie staining: 0.1% (w/v) Coomassie® Brilliant-Blue R-250, 40% Methanol, 10% Acetic acid; Store at room temperature.
· Buffer for Trypsin digestion: 10 mM NH4HCO3, pH 8.5; Prepare a fresh solution for each experiment.
· Matrix for MALDI: alpha-Cyano-4-Hydroxy-Cinnamic Acid (5 mg/ml) in 50% Acetonitrile/0.2% TFA; Store at room temperature but not longer than 3 days.