2. Materials and Methods
2.1 Materials
2.1.3 Chemicals and reagents
Chemical Company
0.9% NaCl solution B. Braun, Melsungen, Germany
10x reaction buffer for M-MulV New England BioLabs GmbH, Frankfurt am Main, Germany
3,3‘-Diaminobenzidin-tetrahydrochloride (DAB) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Acetic acid Carl Roth GmbH & Co. KG, Karlsruhe, Germany Adenosine 5‘-triphosphate (ATP)
disodium salt hydrate Sigma-Aldrich Co., St. Louis, USA
Agarose GeneOn GmbH, Ludwigshafen am Rhein, Germany
Albumin Fraction V (BSA) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Ammonium persulfate (APS) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Ammonium sulfate (NH4)2SO4 Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Aprotinin Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Boric acid Carl Roth GmbH & Co. KG, Karlsruhe, Germany
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Bromophenol blue Sigma-Aldrich Co., St. Louis, USA
Butella Sunflower Oil Brökelmann + Co, Oelmühle GmbH + Co, Hamm, Germany
Chloroform Merck Millipore, Darmstadt, Germany
Citric acid monohydrate Merck Millipore, Darmstadt, Germany
Coelenterazine Promega, Madison, USA
Coenzyme A (CoA) Sigma-Aldrich Co., St. Louis, USA Crystal violet Merck Millipore, Darmstadt, Germany Deoxycholic acid (DOC) AppliChem GmbH, Darmstadt , Germany Deoxynucleotides (dNTPs) Jena Bioscience GmbH, Jena, Germany Dextrane Sulfate Sodium Salt, reagent
grade MO Biomedicals, LLC, Illkirch, France
Diethylpyrocarbonate (DEPC) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Dimethyl sulfoxide (DMSO) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Dimethylformamide Carl Roth GmbH & Co. KG, Karlsruhe, Germany Dipotassium phosphate (K2HPO4) Carl Roth GmbH & Co. KG, Karlsruhe, Germany DL-Dithiothreitol (DTT), BioUltra,
≥99.0% Sigma-Aldrich Co., St. Louis, USA
Eosin Y solution, 1% in water Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Ethanol Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Ethylene diamine tetraacetic acid
(EDTA) Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Ethylene diamine tetraacetic acid
disodium salt dihydrate (Na2EDTA) Carl Roth GmbH & Co. KG, Karlsruhe, Germany ExtrAvidin-Peroxidase Sigma-Aldrich Co., St. Louis, USA
Ethylene glycol tetraacetic acid (EGTA) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Formaldehyde solution 35%, DAB, for
histology Carl Roth GmbH & Co. KG, Karlsruhe, Germany
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Gene RulerTM DNA-Ladder Fermentas GmbH, St. Leon-Rot, Germany Glutaraldehyde, 25% EM Grade
Aqueous Sigma-Aldrich Co., St. Louis, USA
Glycerol Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Glycine Carl Roth GmbH & Co. KG, Karlsruhe, Germany
GlycylGlycine Sigma-Aldrich Co., St. Louis, USA
Guaiac resin Carl Roth GmbH & Co. KG, Karlsruhe, Germany HD Green® DNA stain Intas Science Imaging GmbH, Göttingen, Germany
HEPES Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Hydrochloric acid (HCl) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Hydrogen peroxide solution (H2O2),
30% Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Iodoacetamide Sigma-Aldrich Co., St. Louis, USA
Isopropanol Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Leupeptin Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Lipofectamine® 2000 Invitrogen GmbH, Karlsruhe , Germany Lipofectamine® RNAiMAX Invitrogen GmbH, Karlsruhe , Germany
Magnesium chloride (MgCl2) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Magnesium sulfate (MgSO4) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Mayer‘s haematoxylin solution Merck Millipore, Darmstadt, Germany
Methanol Carl Roth GmbH & Co. KG, Karlsruhe, Germany
N-ethylmaleimide (NEM) Sigma-Aldrich Co., St. Louis, USA Nonidet® P 40 Substitute (NP-40) Sigma-Aldrich Co., St. Louis, USA Nuclear fast red-aluminum sulfate
solution 0.1% Carl Roth GmbH & Co. KG, Karlsruhe, Germany PageRulerTM Plus Prestained Protein
Ladder Fermentas GmbH, St. Leon-Rot, Germany
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Paraffin Rotiplast Carl Roth GmbH & Co. KG, Karlsruhe, Germany Paraformaldehyde (PFA) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Passive lysis buffer (5x) Promega, Madison, USA
PBS tablets GIBCO®, Invitrogen GmbH, Darmstadt, Germany Pefabloc SC Protease Inhibitor Carl Roth GmbH & Co. KG, Karlsruhe, Germany pH solutions (pH 4.01, 7.01, 10.01) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Ponceau S solution Sigma-Aldrich Co., St. Louis, USA
Potassium chloride (KCl) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Potassium ferricyanide (III)
medium Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Rotiphorese® Gel 30 Carl Roth GmbH & Co. KG, Karlsruhe, Germany Salmon sperm DNA Stratagene, La Jolla, USA
Powdered milk Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Sodium azide Sigma-Aldrich Co., St. Louis, USA
Sodium chloride (NaCl) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Sodium deoxycholate AppliChem GmbH, Darmstadt, Germany
Sodium dodecyl sulfate (SDS) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Sodium hydroxide (NaOH) Carl Roth GmbH & Co. KG, Karlsruhe, Germany
SYBR Green I Roche Diagnostics GmbH, Mannheim , Germany
Tamoxifen (>99%) Sigma-Aldrich Co., St. Louis, USA
Temgesic® RB Pharmaceuticals Limited, Berkshire, UK
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Tetramethylethylenediamine (TEMED) Carl Roth GmbH & Co. KG, Karlsruhe, Germany Tissue-Tek O.C.TTM Compound Sakura Finetek USA, Inc., Torrance, USA Tris(hydroxymethyl)aminomethane Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Triton X-100 AppliChem GmbH, Darmstadt , Germany
Tri-sodium citrate Carl Roth GmbH & Co. KG, Karlsruhe, Germany TRIzol® Reagent Invitrogen GmbH, Karlsruhe , Germany
Tween-20 Carl Roth GmbH & Co. KG, Karlsruhe, Germany
Xylol (Isomere) Carl Roth GmbH & Co. KG, Karlsruhe, Germany
X-β-Gal Carl Roth GmbH & Co. KG, Karlsruhe, Germany
α,α-Trehalose dihydrate AppliChem GmbH, Darmstadt , Germany β-Glycerolphosphate (BGP) Sigma-Aldrich Co., St. Louis, USA
Table 4: Enzymes.
Enzyme Company
M-MuLV Reverse Transcriptase (25 U) New England BioLabs GmbH, Frankfurt am Main, Germany
Proteinase K LifeTechnology, Carlsbad, USA
RNase inhibitor New England BioLabs GmbH, Frankfurt am Main, Germany
Taq-Polymerase (5 U/µl) Primetech, Minsk, Belarus
Table 5: Inhibitors.
Inhibitor Cat. no. Source
JQ1 (C23H25ClN4O2S) Stefan Knapp, Goethe-University Frankfurt am Main, Germany
Ganetespib S1159 Selleckchem
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MG-132 S2619 Selleckchem