The tibia was thoroughly flushed bone marrow cells with saline. The flushed tibia was homogenized using a handy sonicator and then centrifuged at 12,000 × g for 30 minutes. The supernatant was subsequently assayed for the protein concentration using the DC Protein Assay Kit (BioRad, Hercules, CA). Equal amounts (20 μg) of total protein were subjected to 10% SDS-PAGE and electroblotted onto a PVDF membrane. The membranes were blocked in 2% blocking reagent (ECL Advance Blocking Agent, GE Healthcare) and probed with antibodies against Cx43 (1:1,000; Cell signaling), RANKL (1:1,000; abcam), SOD2 (1:2,000; SOD-110; StressGen), Sclerostin (1:500; abcam), p-ERK, ERK (1:1,000; Cell signaling), GAPDH (1:1,000; Cell signaling) and ACTIN (1:2,000; Cell signaling). The signals were detected using the ECL system (ECL plus, GE Healthcare) and a luminoimage analyzer LAS-3000 mini (Fuji Film, Tokyo, Japan).
Sod 110
Manufactured by Stressgen
Sourced in Canada, United States
The SOD-110 is a laboratory instrument designed for the measurement of superoxide dismutase (SOD) activity. It utilizes a spectrophotometric method to determine the level of SOD in a given sample. The core function of the SOD-110 is to provide a quantitative assessment of SOD levels, which is a key indicator of oxidative stress in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Sod 101, by Stressgen (3 mentions)
Sc 30147, by Santa Cruz Biotechnology (2 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Ab8227, by Abcam (2 mentions)
Sclerostin, by Abcam (1 mentions)
Ecl advance blocking agent, by GE Healthcare (1 mentions)
P erk erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Las 3000 mini, by Fujifilm (1 mentions)
Rankl, by Abcam (1 mentions)
Ab6201, by Abcam (1 mentions)
Sc 32886, by Santa Cruz Biotechnology (1 mentions)
Ecl film, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Sab4501984, by Merck Group (1 mentions)
5 protocols using sod 110
1
Bone Marrow Protein Profiling
2
Antibody Panel for Mitochondrial Proteins
3
Protein analysis by Western blot
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Tissue homogenizations in 0.05 M sodium phosphate buffer (pH 6.65) were described in our earlier research (Gavrilović et al., 2012 (link)). The protein concentration was measured by BCA method (Thermo Scientific Pierce, Waltham, MA, USA), described by Stich (1990) (link). Determination of SOD1, SOD2, CAT and GPx was performed using Western blot analysis. Specifically, antibodies used for quantification of proteins were for SOD1 (dilution 1:2,000; SOD-101; Stressgen, Canada), for SOD2 (dilution 1:2,000; SOD-110; Stressgen, Canada), for CAT (dilution 1:2,000; Calbiochem, Germany), GPx (dilution 1:500; sc-30147 Santa CruzBiotechnology, Dallas, TX, USA) and for β-actin (dilution 1:1,000; ab8227, Abcam, USA). A secondary antibody (anti-rabbit, dilution 1:5,000; Amersham ECL™ Western Blotting Analysis System; Amersham, UK) was then visualized by the Western blotting enhanced chemiluminescent detection system (ECL; Amersham Biosciences, Amersham, UK). The result was expressed in arbitrary units normalized in relation to β actin, following our previous protocol (Gavrilović et al., 2013 (link)).
4
Hippocampal Protein Quantification
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The hippocampus was homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using BCA method (Thermo Scientific Pierce, USA), described by Stich [30 (link)]. CuZn SOD (SOD1), Mn SOD (SOD2), CAT, GPx, GR, BDNF, TH, DAT, and COMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [27 (link)]. Antibodies used for the quantification of specific proteins were as follows: SOD1 (SOD-101, Stressgen, USA), SOD2 (SOD-110, Stressgen, USA), CAT (Calbiochem, Germany), GPx (sc-30147 Santa Cruz Biotechnology, USA), GR (sc-32886, Santa Cruz Biotechnology, USA), BDNF (ab6201, Abcam, USA), TH (ab51191, Abcam, USA), DAT (ab18548, Abcam, USA), and β-actin (ab8227, Abcam, USA). After washing, the membranes were incubated in the secondary anti-rabbit (dilution 1 : 5000, Amersham ECL™ Western Blotting Analysis System, UK) antibodies conjugated to horseradish peroxidase. A secondary antibody was then visualized by the Western blotting-enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK). The membranes were exposed to ECL film (Amersham Biosciences, UK). The result was expressed in arbitrary units normalized in relation to β-actin, which is in accordance with our previous protocol [27 (link)].
5
Multiplex Analysis of Insulin Signaling
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High throughput xMAP technology (Magpix; Luminex Corporation) was used to asses cytokine profile and protein phosphorylation at insulin receptor (IR Y1162/Y1163 ), insulin receptor substrate-1 (IRS-1 S312 ), protein kinase-B (AKT S473 ), glycogen synthase kinase-3β (GSK3β S9 ), mammalian target of rapamycin (mTOR S2448 ), proline-rich AKT substrate protein (PRAS40 T246 ), ribosomal protein-S6 kinase (P70S6K T421/ S424 ), p38/SAPK Thr180/Tyr182 and mitogen-activated protein kinase/ extracellular signal-regulated kinase-1/2 (MAPK/ERK1/2 Thr185/Tyr187 ) levels, using commercial kits (Life Technologies, USA) [4] . Protein phosphorylation levels are expressed as phospho-protein units per total pg of the same protein. Cytokine levels were normalized by protein content. Protein levels of NRF2 and PGC1α (SAB4501984 and SAB21087, Sigma-Aldrich), mitochondrial dynamin like GTPase (OPA1) and dynamin-related protein-1 (DRP1; 5391 and 80471, Cell Signaling Technology), superoxide dismutases Mn SOD and Cu/Zn SOD (SOD-110 and SOD-101, Stressgen), were measured by western blot.
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