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A user with 403 edits. Account created on 13 February 2025.
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27 February 2025

  • 13:3913:39, 27 February 2025 diff hist −20 Luciferase Reporter Assay No edit summary current Tag: Visual edit
  • 13:3813:38, 27 February 2025 diff hist +1,970 N Luciferase Reporter Assay Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Ice bath for buffers, Luciferin and lysates PBS rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C) Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C) Luciferin aliquots (-20°C freezer) Multipipette and plastic box for solution (cell culture room) | | |- |Assay buffer (100ml): |25 mM Tris pH 7.8 [1 M] → 2.5 ml..." Tag: Visual edit
  • 13:3513:35, 27 February 2025 diff hist +1,996 N Granulocyte oxidative burst Created page with "{| class="wikitable" |+ !Materials !and Preparations |- | |2 days before experiment, streak out gonococci and select the colony phenotype one day before experiment. |- | |Chemiluminescense buffer (CL-Buffer): 8 g NaCl; 0.2 g KCl; 0.62 g KH<sub>2</sub>PO<sub>4</sub>; 1.14 g Na<sub>2</sub>HPO<sub>4</sub>; 2.5 ml 40% glucose; 50 mg BSA; solve in 800 ml bidest. H<sub>2</sub>O; adjust pH to 7.2 and fill it up to 1 liter; steril filter and store at 4°C. |- | |0.2 mg/ml PMA (P..." current Tag: Visual edit
  • 13:3113:31, 27 February 2025 diff hist +2,663 N StrepTactin-Pulldown Created page with "{| class="wikitable" |+ !Materials ! |- |Recombinant protein |Bait protein TwinStrepTagII tagged protein, target proteins (HisSUMO or GST tagged protein of interest) |- |Pull down buffer |50 mM Tris/Cl pH 8, 150 mM NaCl, 10% Glycerol, 0.05% Tween. Optional: 10 µM ZnCl<sub>2</sub> in case of Zinc- finger proteins or 2 mM MgCl<sub>2</sub>, CaCl<sub>2</sub> or MnCl<sub>2</sub> in case of metalloproteins) |- |Elution buffer EXT |50 mM Tris/Cl pH 8, 150 mM NaCl, 50 mM Biotin..." Tag: Visual edit
  • 13:2713:27, 27 February 2025 diff hist +1,746 N Rac pulldown assay (with GST-PBD beads) Created page with "{| class="wikitable" |+ !Materials ! !''add fresh'' |- |Lysis Buffer |Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10% | rowspan="2" |DTT 1 mM, Leupeptin, Aprotinin, PMSF |- |Wash Buffer |Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl<sub>2</sub> 30 mM, NP40 1% |- | |Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD) | |} === Procedure === ---- * Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice. * Stimu..." current Tag: Visual edit
  • 13:1613:16, 27 February 2025 diff hist +2,554 N GST-Pulldown Created page with "{| class="wikitable" |+ !Materials ! |- |GST-Lysis Buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment) |- |GST-Pulldown/wash buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment) |- | |Purified recombinant..." current Tag: Visual edit
  • 13:1213:12, 27 February 2025 diff hist +880 N Fibronectin-Binding Assay Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |humanes Serum (500µl/Probe) | | |- | |gekoppelte Latex Beads | | |- | |α-Fn Antikörper (HFN7.1) | | |- | |Zweitantikörper (α-MCy2) | | |} === '''Durchführung''' === ----Von den zu untersuchenden Beads werden 30-60µl mit 500µl humanem Serum gemischt und 2h bei RT auf dem Rotator inkubiert. Anschliessend werden die Proben zweimal mit PBS gewaschen und mit □-Fn Antikörper versetzt (Zellkulturüberstand unverdünnt)...." current Tag: Visual edit
  • 13:1113:11, 27 February 2025 diff hist +2,437 N Covalent coupling of proteins to latex beads Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |Borate buffer |0.2 M of boric acid with 1 M NaOH, adjusted to a pH value of 8.5 | | |- |2% Carbodiimide solution |2% 1-(3-Dimethylaminopropy)-3-ethyl-carbodiimide hydrochloride are dissolved in MES buffer. Prepare only 15 minutes before use | | |- |Carbonate buffer |0.1 M Na<sub>2</sub>CO<sub>3</sub> are added to 0.1 M NaHCO<sub>3</sub> until a pH value of 9.6 is reached | | |- |MES buffer |0.1 M MES in water, pH value of 5.2-..." current Tag: Visual edit
  • 13:0813:08, 27 February 2025 diff hist +957 N In vitro Tyrosine Kinase Activity Assay Created page with "{| class="wikitable" |+ !Materials ! |- | |Kinase buffer (125 mM NaCl; 48 mM MgCl<sub>2</sub>; 50 mM HEPES (pH 7.5) |- | |4x SDS Buffer |- | |1 mM ATP |- | |Purified Kinase (e.g. Src or FAK) |- | |Kinase substrate (e.g. CEACAM3-cytoplasmic domain or FAK substrate) |} === Procedure === ---- * 0.2 μg/ml FAK-substrate or 25 ng/μl TwinStrep-Ceacam3-cytoplasmic domains * 0.2 μg/ml hFAK KD (recombinant human FAK kinase domain) or 10 ng/μl Src * with or without (control)..." current Tag: Visual edit
  • 13:0613:06, 27 February 2025 diff hist +3,362 N Protein Phosphatase Assay Created page with "{| class="wikitable" |+ !Materials ! |- | |black flat-bottom 384-well plates (Greiner Bio-One, Germany) |- | |phosphatase buffer (50 mM TrisHCl, pH = 8.0 at 25°C, 200 mM NaCl, 20 mM MnCl2, 2 mM TCEP, 0.01% Tween). TCEP and Mn<sup>2+</sup> are added freshly; 1L bottle is sterile filtered and stored at 4°C. |- | |4-Methylumbelliferyl phosphate (4-MUP) (Sigma Aldrich, Germany) or DiFMUP (ABCR GmbH) is dissolved in DMSO (10 mM stock solution) and stored at -50°C |- | |rec..." current Tag: Visual edit
  • 13:0513:05, 27 February 2025 diff hist +92 N File:Example384wellplate.png No edit summary current
  • 12:4312:43, 27 February 2025 diff hist +2,219 N Determination of Integrin Activity (ELISA-based) Created page with "{| class="wikitable" |+ !Materials ! ! ! |- |'''Wash solution''' |1x PBS mit .0.0 5% BSA | | |- |'''Block solution''' |2% BSA in 1 xPBS | | |- |'''Permeabilization solution''' |0.1% Triton X-100 (0.1% in PBS) | | |- |'''Buffer for antibody''' |0.1% BSA in x PBS | | |- |'''Reaction buffer''' |10 ml Lösung '''B''' + 0.5 ml Lösung '''A''' | | |- |'''Solution A''' |120 mg Tetramethylbenzidin in 5 ml Aceton + 45ml EtOH add 100µl H<sub>2</sub>O<sub>2</sub> | | |- |'''Soluti..." current Tag: Visual edit
  • 12:3612:36, 27 February 2025 diff hist +2,394 N Cell Adherence assay Created page with "'''Materials''' {| class="wikitable" |+ !Materials ! |- | |96 well plates; 15 ml und 30 ml Falcon-tubes |- | |coating solution for wells e.g. collagen type 1 (25 µg/ml) |- | |Soybean Trypsin/Inhibitor (12.5 mg/50 ml) in Suspensionen medium- sterile |- | |Crystal violet (250 mg/ 5 ml 96% Ethanol stock solution) |- | |Suspension medium (DMEM +0,25%BSA) sterile |- | |diluted Crystal violet 1:100 in 0.1M Borat buffer pH: 9.2 (freshly prepared!) |- | |4% PFA (Paraformaldehyd..." current Tag: Visual edit
  • 12:3212:32, 27 February 2025 diff hist +46 Methods and Protocols No edit summary Tag: Visual edit
  • 12:2812:28, 27 February 2025 diff hist +1,103 N Cell viability (MTT Assay) Created page with "===== Solutions ===== MTT-Solution: 5mg/mL (≙12mM) (toxic and mutagenic) solved in PBS, sterile filtered with 0,2 µM filter Formazan solving solution: isopropanol === Procedure === ----Coat plate before usage For 96 well plates: use 1 - 2x104 cells in 100 µL medium per well and treat the cells according to your experimental setup. The cell number above was optimized for 1 day treatment; for longer incubation periods use fewer cells accordingly. * After treatme..." current Tag: Visual edit
  • 12:2612:26, 27 February 2025 diff hist +4 Methods and Protocols No edit summary Tag: Visual edit

15 February 2025

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