Computational protocol: The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin

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Protocol publication

[…] Purified recombinant FLAG tagged mouse adiponectin was fractionated on SDS-PAGE, and the band that corresponded to adiponectin on the Coomassie blue-stained gel was excised. The gel pieces were destained with three 5 min wash cycles using destaining buffer (50 mM ammoniumbicarbonate in 40% (v/v) acetonitrile/water), followed by incubation for 30 min at room temperature with 20 mM DTT in the same buffer, and subsequent addition of iodoacetamide to 40 mM. After incubation for 30 min at room temperature, the gel pieces were washed once with destaining buffer and twice with water, before dehydration with acetonitrile and drying in a speed vac device. The dried gel pieces were rehydrated with trypsin solution (5 ng/µl in 25 mM ammoniumbicarbonate 10/90% (v/v) acetonitrile water) stored on ice for about 30 min and then incubated over night at 37°C. Tryptic peptides were extracted from the gel with 20 µl 30% (v/v) acetonitrile/water by ultrsonication for 10 min. Peptide mass fingerprints were measured on an UltrafleXtreme MALDI Tof mass spectrometer (Bruker Daltonics) in reflector mode. To that end, 0.5 µl, of the peptides solution was spotted on an anchor chip samples stage, dried, and overlaid with α-cyanohydroxicynnamic acid as matrix, following the protocols of the manufacturer. The instrument was tuned for peptide measurements between m/z 700 and 5000, external calibration was achieved with standard peptides including insulin to cover the higher mass range. Protein identification and data analysis was done with the Flex Analysis and Biotools software packages (Bruker Daltonics) using Mascot (Matrix Science) as search engine. [...] In order to analyze the amount of adiponectin, equal volumes of cell culture medium or diluted serum were loaded onto a 15% SDS-PAGE. For the analysis of the molecular weight of adiponectin, 5 µl and 10 µl of diluted (1/300) wild type and LH mutant serum samples, respectively, and equal volumes of cell culture medium were separated near the end of gel. For immunoblot analysis adipose tissue was homogenized with 0.1 M glycine, 0.02 M Tris-HCl pH 7.8, 1% Igepal, or 25 mM Hepes pH 7.5, 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 10% glycerol, 1% Triton X-100 buffer including Complete EDTA-free protease inhibitor cocktail (Roche), and disrupted by brief sonication. The cell debris was removed by centrifugation. The protein concentrations were measured with Protein assay (Bio-Rad), and equal amounts of soluble protein were loaded onto the gel. The proteins were separated under reducing conditions by SDS-PAGE and transferred to an Immobilon-P transfer membrane (Millipore). For analysis of adiponectin oligomers equal volumes of cell culture medium were separated in non-reducing and non-heat-denaturing conditions . The membranes were blocked with 5% milk powder in Tris buffered saline – Tween 20 and incubated with rabbit anti-adiponectin (Affinity BioReagents) followed by a horseradish peroxidase-conjugated anti-rabbit IgG (P.A.R.I.S. Biotech). Immunocomplexes were visualized using an ECL+ detection system (GE Healthcare) and Kodak XAR films or Molecular imager ChemiDoc XRS+ (Bio-Rad). The mobility of the adiponectin monomer was measured using CorelDraw software and size of the monomer was calculated using molecular weight standard curve. Quantification of adiponectin levels was performed with ImageQuant TL 7.0 (GE Healthcare) or Image Lab software (Bio-Rad). […]

Pipeline specifications

Software tools Biotools, CorelDraw, Imagequant TL, Image Lab
Applications Miscellaneous, Population genetic analysis
Organisms Mus musculus, Homo sapiens
Diseases Metabolic Diseases
Chemicals Cholesterol, Glucose