Tissue samples were homogenized in ice-cold RIPA lysis buffer containing a protease inhibitor. The homogenates were centrifuged at 14 000 × g for 20 min at 4 °C. The lysate protein concentration was determined using the Bradford method (Bio-Rod Laboratories, Munich, Germany). An equal volume of 2X SDS sample buffer was added, and then the mixture was boiled for 5 min. The samples were resolved electrophoretically on a 12.5% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked overnight at 4 °C with 5% nonfat powdered milk in TBST and then incubated for 3 h with a primary antibody (SIRT1 or Klotho, 1:500; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at room temperature. After washing in TBST for three times (10 min each), the blots were incubated with goat anti-mouse IgG secondary antibody (1:10000; Santa Cruz Biotechnology Inc.) for 1 h at room temperature. All antibodies were diluted with blocking buffer. The antibody-antigen complex was detected using Western blot documentation system and analyzed with imageJ analyzing software. β-actin immunoblotting was used as a loading control (27 (link)). Serum concentration of angiotensin II, was determined with the ELISA kit (Ray Biotech, Inc., Norcross, GA, USA)
Klotho
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Klotho is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a protein that functions as a cofactor for various enzymes involved in cellular processes. The core function of Klotho is to regulate the activity of these enzymes, though its specific applications may vary depending on the research context.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Goat anti mouse igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Ecl substrate, by Beyotime (1 mentions)
Ccd camera, by Bio-Techne (1 mentions)
Anti fgf23, by Santa Cruz Biotechnology (1 mentions)
Protein assay kit, by Beyotime (1 mentions)
Phosphor stat3, by Cell Signaling Technology (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg secondary antibody, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
4 protocols using klotho
1
Western Blot Analysis of SIRT1 and Klotho
2
Western Blot Analysis of FGF23 and Klotho
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Tissue was first homogenized on ice and centrifuged at 13000 g for 10 min at 4 °C. Superna-tants were collected and protein concentration was determined using Protein Assay Kit (Beyotime). Equal amount of samples were then isolated by 12% SDS-PAGE and transferred onto a 0.45 μm PVDF membrane (Millipore). Subsequently, membrane was first blocked with 5% non-fat milk for 1 hr at room temperature and then incubated with primary antibodies and corresponding HRP-conjugated secondary antibodies for 2 hr and 1hr at room temperature, respectively. After incubation, the membrane was extensively washed and immune-bands on the membrane were visualized using ECL substrate (Beyotime) under a CCD camera (Alpha Innotech). The gray-scale of the bands was analyzed by Quantity-one v4.62 software. The relative expression of FGF23 and Klotho was normalized to that of the internal control GAPDH. Primary antibodies anti-FGF23, Klotho and GAPDH were all purchased from Santa Cruz. HRP-conjugated secondary antibodies were purchased from Boster.
3
Western Blot Analysis of Protein Expression
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Protein level was measured by western blot. Briefly, equal amounts of protein (10μg per lane) were separated by SDS-PAGE electrophoresis, and transferred to PVDF membrane (Bio-Rad Inc.). The membranes were incubated in blocking buffer (0.2 mM Tris, 137 mM NaCl, 5% no-fat milk, and 0.1% Tween-20) for an hour and then probed at 4℃overnight with relevant antibody. The membranes were rinsed with washing buffer (0.1% Tween 20, 0.2 mM Tris, and 137 mM NaCl) and incubated with HRP-conjugated secondary antibody for an hour at room temperature, followed by chemiluminescent detection. Antibodies against Klotho (Santa Cruz), GAPDH, phosphor-STAT3, total STAT3, P53, α-SMA and E-cadherin (all purchased from Cell Signaling) were used to probe the blot.
4
Protein Expression Analysis by Western Blot
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Proteins were separated on SDS-polyacrylamide minigels by electrophoresis [19] (link). After transfer by electroelution to polyvinylidene difluoride (PVDF) membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 60 minutes with 5% non-fat dry milk in Tris-buffered saline solution. Blots were then incubated overnight with antibodies against Actin (1∶5,000), VDR (1∶500), Klotho (1∶500) and TGF-beta (1∶200) (Santa Cruz Biotechnology, CA, USA). The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, diluted 1∶2,000, or anti-goat, diluted 1∶10,000, Sigma Chemical, St. Louis, MO, USA) and enhanced chemiluminescence (ECL) detection system (GE Healthcare Limited, Little Chalfont, UK).
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