Protein preparation, SDS-PAGE, and western blotting were performed as described 27 (link). Briefly, adipocytes treated with butein or siRNAs were harvested and lysed in RIPA buffer (1 % NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 10 mM NaF) containing a protease inhibitor cocktail (Roche Diagnostics). For tissue homogenates, white adipose tissues (100mg) were ground in liquid nitrogen and homogenized in RIPA buffer (200μl) supplemented with protease inhibitors (Roche Diagnostics). Homogenates were centrifuged for 10 min at 14,000 rpm at 4°C and supernatants collected. Protein lysates were separated on SDS-PAGE, transferred to PVDF membranes (Bio-Rad Laboratories), and western blot analysis was performed as described 27 (link). The membranes were blocked for 1 hour with 5% non-fat dry milk and incubated overnight at 4°C with primary antibodies against Prdm4 (1:2,000, ab156867, Abcam), Ucp1 (1:1,000, ab10983, Abcam), or actin (1:5,000, sc47778, Santa Cruz Biotech). The membranes were then probed with HRP-conjugated secondary antibodies (1:10,000, Ab Frontier) and developed by an enhanced chemiluminescent western blotting detection reagent (GE health care).
Enhanced chemiluminescent western blotting detection reagent
Manufactured by GE Healthcare
Sourced in China, United States
Enhanced chemiluminescent western blotting detection reagents are laboratory equipment used to detect and quantify specific proteins in biological samples. These reagents generate a luminescent signal when exposed to the target proteins, allowing for their visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Ab156867, by Abcam (2 mentions)
Hrp conjugated secondary antibodies, by AbFrontier (2 mentions)
Ab10983, by Abcam (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Sc 8334, by Santa Cruz Biotechnology (1 mentions)
Abs1064, by Merck Group (1 mentions)
A5441, by Merck Group (1 mentions)
Anti vegfr2, by Santa Cruz Biotechnology (1 mentions)
Anti vegf a, by Santa Cruz Biotechnology (1 mentions)
Anti vegfr 1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Anti vegfr3, by Santa Cruz Biotechnology (1 mentions)
Anti vegf c, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Pall Corporation (1 mentions)
Anti sirt1, by Merck Group (1 mentions)
6 protocols using enhanced chemiluminescent western blotting detection reagent
1
Protein Analysis in Adipocytes
2
Protein Analysis in Adipocytes
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Protein preparation, SDS-PAGE, and western blotting were performed as described 27 (link). Briefly, adipocytes treated with butein or siRNAs were harvested and lysed in RIPA buffer (1 % NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 10 mM NaF) containing a protease inhibitor cocktail (Roche Diagnostics). For tissue homogenates, white adipose tissues (100mg) were ground in liquid nitrogen and homogenized in RIPA buffer (200μl) supplemented with protease inhibitors (Roche Diagnostics). Homogenates were centrifuged for 10 min at 14,000 rpm at 4°C and supernatants collected. Protein lysates were separated on SDS-PAGE, transferred to PVDF membranes (Bio-Rad Laboratories), and western blot analysis was performed as described 27 (link). The membranes were blocked for 1 hour with 5% non-fat dry milk and incubated overnight at 4°C with primary antibodies against Prdm4 (1:2,000, ab156867, Abcam), Ucp1 (1:1,000, ab10983, Abcam), or actin (1:5,000, sc47778, Santa Cruz Biotech). The membranes were then probed with HRP-conjugated secondary antibodies (1:10,000, Ab Frontier) and developed by an enhanced chemiluminescent western blotting detection reagent (GE health care).
3
Western Blot Analysis of Cellular Proteins
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Cells were washed with ice-cold PBS, then scraped from the surface of the culturing dishes and collected by centrifugation. Total protein lysates were extracted by RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Then, the Bradford assay was used to estimate protein concentrations. Protein samples were loaded onto 10% SDS-PAGE gels and transferred to the PVDF membranes. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline containing 0.05% Tween 20 (TBST) for 1.5 h and then incubated overnight with the primary antibodies at 4 °C. Antibodies against GFP (1:1000, sc-8334, Santa Cruz, CA, USA), hypusine (1:1000, ABS1064, Millipore, Temecula, CA, USA), and β-actin (1:20000, A5441, Sigma-Aldrich) were used in our study. On the next day, after washing three times with 1×TBST, membranes were incubated for 1.5 h with secondary antibodies at room temperature. Protein signals were visualized by Enhanced Chemiluminescent Western Blotting Detection Reagents (GE Healthcare) under a Chemiluminescent Imaging System (Tanon 5200, Tanon Corporation, Minhang, Shanghai, China). The full membranes are shown in Figures S3 and S4 .
4
VEGF Signaling Pathway Protein Detection
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Cells washed with PBS were lysed using radioimmunoprecipitation assay buffer (Pierce, Rockford, IL) that was supplemented with Roche complete protease inhibitor cocktail tablets (Nutley, NJ). Protein extracts were collected via centrifugation at 14,000g for 20 min and protein concentrations were determined with an RC/DC Bio‐Rad assay kit (Bio‐Rad, Hercules, CA) following the manufacturer‐supplied protocol. Protein extracts were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (PALL Corp., Port Washington, NY). The membranes were preincubated with blocking solution (3% (w/v) skim milk in TBS containing 0.1% Tween‐20) for 1 h, incubated with anti‐VEGF‐A, anti‐VEGF‐C, anti‐VEGFR‐1, anti‐VEGFR‐2, anti‐VEGFR‐3 (1:2000 dilution in blocking solution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti‐β‐actin (1:5000 dilution in blocking solution; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4°C, then probed with peroxidase‐conjugated anti‐mouse IgG, anti‐goat IgG, or anti‐rabbit IgG (1:5000 dilution in blocking solution; Santa Cruz Biotechnology, Inc.). Protein bands were detected using enhanced chemiluminescent western blotting detection reagents (GE Healthcare, Piscataway, NJ).
5
Western Blot Analysis of Melanin Proteins
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Protein samples that were collected during the melanin content assay was used for western blot analysis. The lysates were denatured in SDS-PAGE protein loading buffer 5X (cat. no. AR1112; Wuhan Boster Biological Technology, Ltd.) separated on 10% SDS-PAGE at 80 V, and transferred onto polyvinylidene fluoride membranes for 2 h at 400 A. Membrane blocking was performed with 5% skim milk dissolved in TBS with 1% Tween-20 (TBST) at room temperature for 1 h and the membrane was incubated with primary antibodies at dilutions of 1:1,000 at 4°C overnight. Subsequent to washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:2,000 for 1 h at room temperature. The membranes were then washed with TBST. Proteins were visualized by enhanced chemiluminescent western blotting detection reagents (GE Healthcare, Chicago, IL, USA). Densitometry analysis was performed using Quantity One version 3 (Bio-Rad Laboratories, Inc.).
6
Protein Lysate Extraction and Western Blot Analysis
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Protein lysate was extracted with RIPA (10mM Tris-HCl, pH 8, 140mM NaCl, 5mM EDTA, 0.025% NaN3, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with a protease inhibitor cocktail (Roche, 11873580001). The lysates were separated by SDS polyacrylamide gel electrophoresis, and the proteins were transferred to a polyvinylidene difluoride membrane. Anti-Sirt1 (Sigma-Aldrich, 07–131), anti-α-tubulin (Cell signaling technology, 2125), Acetyl-p53 (Lys379) Antibody (Cell Signaling Technology, 2579), p53 (DO-1) (Santa Cruz Biotechnology, sc-126) and Ucp1 (Abcam, ab10983) were used as primary antibodies at a concentration of 1:1000, and 5% skim milk was used for blocking. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G (Jackson, 115-035-144) and HRP AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, 115-035-003) were used as the secondary antibody at a concentration of 1:5000. The proteins were detected by enhanced chemiluminescent Western blotting detection reagents (GE Healthcare). As a positive control for acetyl-p53, 400 nM trichostatin A and 0.5 μM doxorubicin were reacted with HUVECs for 24 h.
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