Proteins were isolated from tissues with lysis buffer [100 mM tris-HCl (pH 6.8), 2.0% SDS, 20% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol, 100 mM NaF, and 1 mM Na3VO4]. The concentration of homogenized protein lysates was determined by the Bradford method (Bio-Rad). The following antibodies were used for the detection of target proteins: UCP1 (#14670), phospho-PKA (#5661), total PKA (#4782), phospho-AMPK (#2535), and total AMPK (#2793) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against glucose transporter 4 (GLUT4; sc-1607), apelin (sc-293441), and HIF-1α (sc-13515) were purchased from Santa Cruz Biotechnology (Dallas, TX). PRDM16 (PA5-20872; Thermo Fisher Scientific, Rockford, IL) and PGC-1α (no. 66369-1-Ig; Proteintech, Rosemont, IL) were also purchased. Antibodies against β-actin and β-tubulin were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). IRDye 680 goat anti-mouse secondary antibody (1:10,000), IRDye 800CW donkey anti-rabbit secondary antibody (1:10,000), and IRDye 800CW donkey anti-goat secondary antibody (1:10,000) were purchased from LI-COR Biosciences (Lincoln, NE). The target proteins were detected using the infrared imaging system (Odyssey, LI-COR Biosciences), and intensity of band was quantified using Image Studio Lite (LI-COR Biosciences) (16 (link)).
Irdye 800cw donkey anti goat secondary antibody
Manufactured by LI COR
The IRDye 800CW donkey anti-goat secondary antibody is a fluorescently labeled antibody that can be used to detect and quantify goat primary antibodies in various immunoassays and imaging applications. The antibody is conjugated with the near-infrared dye IRDye 800CW, which provides strong infrared fluorescence signal for sensitive detection.
Automatically generated - may contain errors
Lab products found in correlation
Image studio lite, by LI COR (2 mentions)
Total ampk, by Cell Signaling Technology (1 mentions)
Irdye 680 goat anti mouse secondary antibody, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit secondary antibody, by LI COR (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Phospho ampk, by Cell Signaling Technology (1 mentions)
Sc 13515, by Santa Cruz Biotechnology (1 mentions)
Total pka, by Cell Signaling Technology (1 mentions)
Odyssey system, by LI COR (1 mentions)
Rock2, by Santa Cruz Biotechnology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Chameleon duo, by LI COR (1 mentions)
Goat anti dnp primary antibody, by Fortis Life Sciences (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
4 protocols using irdye 800cw donkey anti goat secondary antibody
1
Western Blot Analysis of Metabolic Proteins
2
Detecting Sup35 Amyloids by SDD-AGE
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Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was performed based on previously published protocol47 (link). Cells were resuspended in PEB buffer (25 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 2 × PIC, pH 7.5) and lysed by glass beating with the Precellys®24 device (Bertin Corp) at 4 °C. The lysates were precleared by centrifugation at 300 g at 4 °C for 15 min and supernatant was collected. 100 µg of proteins in 4 × Sample Buffer (2 × TAE, 20% glycerol, 8% SDS, 0.03% (w/v) bromophenol blue) were loaded into 1.5% agarose gel in 1 × TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 7.6) containing 0.1% SDS. The gel was run at 120 V at 4 °C until the dye front reached 1 cm from the end of the gel. The proteins were transferred onto nitrocellulose membrane at 25 V at 4 °C for 16 hours. Sup35 monomers and amyloids were subsequently immuno-detected using anti-Sup35 polyclonal antibody (Santa Cruz, sc-25915, 1:1,000 dilution) and the IRDye® 800CW Donkey anti-Goat secondary antibody (LI-COR, 925-32214, 1:20,000 dilution) by standard Western blot and by imaging with the LI-COR Odyssey system.
3
Rho Kinase Quantification in Coronary Arteries
4
Quantifying Protein Carbonylation in Plasma
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Protein carbonylation was determined by derivatization of protein carbonyls with DNPH using a procedure based on previous publications5 (link). Approximately 12 µg of plasma proteins were treated with 6% (w/v) SDS in a 30 µL volume. An equal volume of 20 mM DNPH in 10% (v/v) TFA was added and incubated at room temperature for 10 min. The reaction was neutralized with 30 µL of 2 M Tris in 30% (v/v) glycerol containing 7% (v/v) β-mercaptoethanol, and 15 µL of each DNP-derivatized sample was loaded in two identical gels. One gel was used for Coomassie staining, and proteins from other gel were transferred to an immobilon-P PVDF membrane (Millipore, Billerica, MA). After transfer, the PVDF membrane was immunoblotted with goat anti-DNP primary antibody (Bethyl Laboratories Inc., Montgomery, TX) and donkey anti-goat IRDye 800CW secondary antibody (LI-COR, Lincoln, NE). The DNP-derivatized carbonylated proteins were detected using the Odyssey infrared imaging system (LI-COR, Lincoln, NE).
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