2.1 EQUIPMENT
Centrifuges• Centrifuge 5810R (Eppendorf, Hamburg, Germany)
• Heraeus Fresco 17 Centrifuge (Thermo Scientific, Waltham, MA, USA)
• Heraeus Pico 17 Centrifuge (Thermo Scientific, Waltham, MA, USA)
• Minispin plus (Eppendorf, Hamburg, Germany)
• Avanti Centrifuge J-251, Rotors JA-25.50 and JA-14 (Beckman Coulter, Fullerton, USA)
• Optima LE-80K Ultracentrifuge, Rotor Ti80 (Beckman Coulter, Fullerton, USA) Incubators and shakers
• Incubator for bacterial cultures (Binder, Tuttlingen, Germany)
• Incubator shaker for bacterial cultures Innova 4400 (New Brunswick Scientific, Edison, USA)
• Magnetic stirrer Agimatic-N (JP Selecta, Barcelona, Spain)
• Mini Rocker MR-1 (Biosan, Riga, Latvia)
• Shaker REAX 2 (Heidolph Instruments, Heidelberg, Germany)
• Shaker, horizontal Unimac 1010 (Heidolph Instruments, Heidelberg, Germany)
• Thermomixer comfort (Eppendorf, Hamburg, Germany)
• Ultra Low -80°C freezer (Sanyo, Sakata, Japan)
• Vortex Genius 3 (IKA, Staufen, Germany)
• Water bath (Memmert, Schwabach, Germany) Cell culture
• CO2 incubator (Sanyo, Sakata, Japan)
• CO2 incubator HeraCell (Heraeus/Kendro Laboratory Products, Hanau, Germany)
• Safety cabinet HeraSafe (Heraeus/Kendro Laboratory Products, Hanau, Germany)
• Peristaltic pump Masterflex 701572 (Cole-Parmer, Vernon Hills, USA)
• Pump Laboport (KNF Neuberger, Freiburg, Germany)
• Electro Cell Manipulator (ECM) 630 (BTX Harvard Apparatus, Holliston, MA, USA) Optical equipment and microscopes
• Leica DMIL microscope (Leica, Heidelberg, Germany; cell culture)
• Olympus IX81 microscope; PlanApo 100x/1.40 oil objective (Olympus Optical, Hamburg, Germany)
• Camera F-View II CCD and Soft Imaging software (Soft Imaging Systems, Münster, Germany)
• Zeiss LSM 510 confocal microscope; Plan-Apochromat 63x and 100x/1.4 oil objectives (Carl Zeiss, Oberkochen, Germany)
• Zeiss LSM 510 live cell equipment: Heating insert P, CTI controller 3700 digital, Tempcontrol 37-2 (PeCon GmbH, Erbach, Germany)
• Leica TCS SP2 AOBS confocal microscope; PlanApo 100x/1.40 oil objective (Leica, Wetzlar, Germany)
• Andor Revolution XD spinning disk confocal microscope (Andor technology, Belfast, Northern Ireland)
• AlphaImager HP (Alpha Innotech, San Leandro, USA)
• BioRad Molecular Imager FX (BioRad, Munich, Germany)
• Phospho-Imager FLA-3000 (Fujifilm Europe GmbH, Düsseldorf, Germany)
• Calibrated Imaging Densitometer GS-710 (Bio-Rad, Munich, Germany)
• Photometer Ultrospec 100 pro (Amersham Biosciences, Uppsala, Sweden)
• Qubit fluorometer (Invitrogen, Carlsbad, CA, USA)
• NanoDrop nanospectrophotometer 2000 (Fisher Scientific – NanoDrop products, Wilmington, DE, USA)
• Tecan M200 infinite plate reader (Tecan Group Ltd., Männedorf, Switzerland) Electrophoretic and blotting equipment
• Horizontal PerfectBlue Gel System Mini (Peqlab Biotechnology, Erlangen, Germany)
• Electrophoresis power supply EPS 2A200 (Amersham Biosciences, Uppsala, Sweden)
• Electrophoresis power supply EPS 601 (Amersham Biosciences, Uppsala, Sweden)
• Slap mini gel chamber (Keutz, Reiskirchen, Germany)
• Trans-Blot SD SemiDry transfer cell (BioRad, Munich, Germany) Other equipment
• Autoclave Uniclave 88 (AJC, Lisbon, Portugal)
• Bag Sealer Folio (Severin, Sundern, Germany)
• Balance Vicon (Acculab/Sartorius, Göttingen, Germany)
• Balance, analytical BP 221S (Sartorius, Göttingen, Germany)
• Centrifuge beakers, 250 ml (Beckman, Munich, Germany)
• Centrifuge tubes, 50 ml (Nalgene, Rochester, USA)
• Cryomed N2 storage (Forma Scientific, Waltham, USA)
• Drying Oven (Sanyo, Sakata, Japan)
• Easypet (Eppendorf, Hamburg, Germany)
• Microwave KOR-63A5 (Daewoo, Butzbach, Germany)
• pH meter PB-11 (Sartorius, Göttingen, Germany)
• Research pipettes, 2.5 µl, 10 µl, 100 µl, 1000 µl (Eppendorf, Hamburg, Germany)
• Thermal Cycler MyCycler (Bio-Rad, Munich, Germany)
• X-Ray cassette (Roth, Karlsruhe, Germany)
• Autoradiography cassette BAS Cassette 2040 (Fujifilm Europe GmbH, Düsseldorf, Germany)
• Fujifilm Imaging Plate BAS-MP 2040S (Fujifilm Europe GmbH, Düsseldorf, Germany)
• Ultracentrifuge adapters for reaction tubes (Rotor: 70Ti; Beckman Coulter) (Beranek
2.2 CONSUMABLES
Product Source
Centrifuge tubes, 250 mL Nalgene, Rochester, NY, USA Centrifuge tubes, 50 mL Nalgene, Rochester, NY, USA Cover slips, round, 12 and 18 mm diameter Menzel, Braunschweig, Germany
Cryovials, 2 mL Greiner Bio-One, Frickenhausen, Germany
Cuvettes, semimicro Plastibrand/Brand, Wertheim, Germany Dishes for agar plates Greiner Bio-One, Frickenhausen, Germany Dishes for tissue culture, 35, 60, 100 mm Greiner Bio-One, Frickenhausen, Germany Dishes for tissue culture, 6, 12 and 24 well Greiner Bio-One, Frickenhausen, Germany Dishes for tissue culture, 96 well, black well,
flat, microclear bottom
Greiner Bio-One, Frickenhausen, Germany Electroporation cuvettes, 4 mm gap Molecular BioProducts, San Diego, USA Filter for medium, Sartolab P20, 0.2 µm Sartorius, Goettingen, Germany
Folded filters Macherey-Nagel, Düren, Germany
Glass bottom dishes, 35 mm MatTek, Ashland, USA
Hyperfilm ECL Amersham Biosciences, Uppsala, Sweden
Microcentrifuge tubes, 0.2 mL Axygen, Union City, CA, USA Microcentrifuge tubes, 0.5 mL for Qubit Axygen, Union City, CA, USA Microcentrifuge tubes, 0.5, 2 mL Sarstedt, Nürnbrecht, Germany
Microcentrifuge tubes, 1.5 mL Greiner Bio-One, Frickenhausen, Germany Microcentrifuge tubes, 1.5mL, SafeLock Eppendorf, Hamburg, Germany
Microscope slides, frosted end Menzel, Braunschweig, Germany Needle, Sterican 20 G x 1”, 0.45 x 25 mm B. Braun, Melsungen, Germany Needle, Sterican 26 G x 1”, 0.45 x 25 mm B. Braun, Melsungen, Germany Pipette tips, 20, 200, 1000 µ L Sarstedt, Nümbrecht, Germany Protran Nitrocellulose Transfer Membrane Whatman, Dassel, Germany
Reaction tubes, 15, 50 mL Greiner Bio-One, Frickenhausen, Germany
Rubber policeman Roth, Karlsruhe, Germany
Serological pipettes, 5, 10 mL Greiner Bio-One, Frickenhausen, Germany
Silica Gel Orange Roth, Karlsruhe, Germany
Syringe Inject, 10 mL B. Braun, Melsungen, Germany
Syringe Omnifix-F, 1 mL B. Braun, Melsungen, Germany
Tubes for bacterial cultures, 14 mL Greiner Bio-One, Frickenhausen, Germany Whatman filter paper, 3 mm Whatman, Dassel, Germany
Table 2.1: Consumables
2.3 CHEMICALS AND REAGENTS 2.3.1 Chemicals
Chemical Source
[P32] orthophosphoric acid PerkinElmer, Waltham, MA, USA
[S35]-methionine Hartmann Analytics, Braunschweig,
Germany 2-7-dichlorodihydrofluorescein diacetate
(H2DCFDA)
Invitrogen, Carlsbad, CA, USA
2-Mercaptoethanol Sigma, Steinheim, Germany
6-hydroxydopamine Sigma, Steinheim, Germany
Chemical Source
Acrylamide – RotiphoreseGel 30 Roth, Karlsruhe, Germany
Agar Formedium, Hunstanton, England
Agarose NEEO Roth, Karlsruhe, Germany
Albumin Fraction V (BSA) Roth, Karlsruhe, Germany Ammonium persulphate (APS) Sigma, Steinheim, Germany
Ampicillin Sigma, Steinheim, Germany
Arachidonic acid MP Biochemicals, Illkirch, France
Bezafibrate Boehringer-Mannheim, Mannheim, Germany
Bromophenol blue Sigma, Steinheim, Germany
Calcium chloride Sigma, Steinheim, Germany
CHAPS Roth, Karlsruhe, Germany
Chloroform Merck, Darmstadt, Germany
Copper Sigma, Steinheim, Germany
Cycloheximide Sigma, Steinheim, Germany
D(+)glucose monohydrate Fluka/Sigma, Steinheim, Germany
Dexamethasone Sigma, Steinheim, Germany
Digitonin Sigma, Steinheim, Germany
Dimethyl sulphoxide (DMSO) Sigma, Steinheim, Germany
Dithiothreitol (DTT) Sigma, Steinheim, Germany
ECL advanced western blot detection kit Amersham Biosciences, Uppsala, Sweden Eicosatetraynoic acid (ETYA) Alexis/Axxora, Grünberg, Germany
Ethanol Merck, Darmstadt, Germany
Ethidium bromide solution Fluka/Sigma, Steinheim, Germany Ethylenediaminetetraacetic acid salt
dehydrate (EDTA)
Sigma, Steinheim, Germany
FOY-305 protease inhibitor Sanol-Schwarz, Monheim, Germany
G418 (Geneticin) Sigma, Steinheim, Germany
Glycerol Roth, Karlsruhe, Germany
Glycine Sigma, Steinheim, Germany
HEPES sodium salt Sigma, Steinheim, Germany
Hoechst 33528 Polysciences, Warrington, USA
Hydrochloric acid Merck, Darmstadt, Germany
Hydrogen peroxide (30%) Merck, Darmstadt, Germany
Isopropanol Merck, Darmstadt, Germany
Kanamycin disulphate salt Sigma, Steinheim, Germany
LB-Broth Miller Formedium, Hunstanton, England
Malonate Sigma, Steinheim, Germany
Menadione Sigma, Steinheim, Germany
Methanol Merck, Darmstadt, Germany
Milk, powder, low fat (Molico) Nestlé, Linda-a-Velha, Portugal
MOPS sodium salt Sigma, Steinheim, Germany
Mowiol 4-88 reagent Calbiochem/Merck, Darmstadt, Germany
Nickel Sigma, Steinheim, Germany
n-propylgallate Fluka/Sigma, Steinheim, Germany
Oleic acid Merck, Darmstadt, Germany
Palmitic acid MP Biochemicals, Illkirch, France
Chemical Source
Polyethyleneglycol (PEG) 6000 Fluka/Sigma, Steinheim, Germany
Ponceau S Roth, Karlsruhe, Germany
Potassium chloride Sigma, Steinheim, Germany
Potassium phosphate dibasic Sigma, Steinheim, Germany Protein A-Sepharose, 1 mg/ml Sigma, Steinheim, Germany Protein assay (Bradford) Bio-Rad, Munich, Germany
Proteinase K Sigma, Steinheim, Germany
Sodium azide Sigma, Steinheim, Germany
Sodium bicarbonate Sigma, Steinheim, Germany
Sodium carbonate Sigma, Steinheim, Germany
Sodium chloride Sigma, Steinheim, Germany
Sodium deoxycholate monohydrate Sigma, Steinheim, Germany Sodium dodecyl sulphate (SDS) Sigma, Steinheim, Germany
Sodium fluoride Sigma, Steinheim, Germany
Sodium hydroxide Merck, Darmstadt, Germany
Sodium orthovanadate Sigma, Steinheim, Germany
Sodium phosphate dibasic Sigma, Steinheim, Germany
Sucrose Sigma, Steinheim, Germany
Sudan Black Merck, Darmstadt, Germany
Tetramethylethelenediamine (TEMED) Fluka/Sigma, Steinheim, Germany Trasylol (protease inhibitor) Bayer, Leverkusen, Germany Trichloroacetic acid (TCA) Merck, Darmstadt, Germany
Tris Roth, Karlsruhe, Germany
Triton X-100 Sigma, Steinheim, Germany
Water, treated with DEPC Roth, Karlsruhe, Germany
Table 2.2: Frequently used chemicals
Further chemicals not listed in Table 2.2 were purchased from Sigma (Steinheim, Germany), Merck (Darmstadt, Germany), or Roth (Karlsruhe, Germany).
2.3.2 Loading dyes and markers
Product Source
6x Orange Loading Dye Fermentas, Burlington, Canada Gene Ruler DNA Ladder Mix Fermentas, Burlington, Canada Kaleidoscope Precision Plus Protein
Standard
BioRad, Munich, Germany Precision Plus Proteins Standard BioRad, Munich, Germany
Table 2.3: Loading dyes and markers
2.3.3 Kits
Product Source
Nucleobond Xtra Midi Kit Macherey-Nagel, Düren, Germany Qiaprep Spin Miniprep Kit Qiagen, Hilden, Germany
Qiaquick Gel Extraction Kit Qiagen, Hilden, Germany Quant-iT dsDNA BR Assay Kit Invitrogen, Carlsbad, CA, USA Quant-iT RNA Assay Kit Invitrogen, Carlsbad, CA, USA RNeasy Protect Mini Kit Qiagen, Hilden, Germany
Table 2.4: Kits
2.3.4 Cell culture reagents
Product Source
Chloroquine diphosphate Fluka/Sigma, Steinheim, Germany Collagen R solution (4mg/mL) Serva, Heidelberg, Germany DEAE-Dextran hydrochloride Sigma, Steinheim, Germany
DMEM high glucose with L-glutamine PAA Laboratories, Pasching, Austria DMEM with L-glutamine, without sodium
bicarbonate and phenol red, powder (supplemented with 3.7 g/l sodium bicarbonate and filtered upon use)
Sigma, Steinheim, Germany
Fetal Bovine Serum “Gold” (FBS) PAA Laboratories, Pasching, Austria Ham’s F-12 with L-glutamine, powder
(supplemented with 1.18 g/L sodium bicarbonate and filtered upon use)
Sigma, Steinheim, Germany
L-glutamine (100x) Sigma, Steinheim, Germany
Lipofectamine reagent Invitrogen, Karlsruhe, Germany MEM essential medium (MEM) without
L-glutamine, without L-methionine
Biowest S.A.S., Nuaille, France N1 medium supplement for serum-free
culture (100x)
Sigma, Steinheim, Germany
Penicillin/Streptomycin, 100x concentrate PAA Laboratories, Pasching, Austria Polyethylenimine (PEI) 25 kD Sigma, Steinheim, Germany
Poly-L-Lysine Sigma, Steinheim, Germany
Trypsin/EDTA PAA Laboratories, Pasching, Austria
Turbofect transfection reagent Fermentas, Burlington, Canada
Table 2.5: Frequently used cell culture reagents
2.4 IMMUNOLOGICAL REAGENTS 2.4.1 Primary antibodies
Antibody Source Dilution
Mouse mc anti-Actin 2G2 B. Jokusch, University of Braunschweig, Germany
WB 1:1000