The protein concentration of the whole epididymal adipose tissue extract (including the cell membrane, cytoplasm and nucleus) was determined by the Bradford method(34 (link)). Samples (25 µg of protein) were heated in Laemmli buffer at 100°C for 5 min, then loaded onto a 10 % SDS–polyacrylamide gel. Transfer to a nitrocellulose membrane was carried out at 4°C in the presence of methanol. Incubation with the primary antibodies (purchased from Santa Cruz Biotechnology) was performed overnight at 4°C in Tris-buffered saline solution containing Tween 20 (TBS-T) and 3 % non-fat dried milk. Antibody dilutions were: 1:200 for mouse anti-TLR-4 sc293072, 1:200 for mouse anti-β-actin sc47778, 1:100 for rabbit anti-phosphorylated NF-κB (ser536) sc33020, and 1:200 for mouse anti-total NF-κB sc8008. After incubation overnight at 4°C in TBS-T containing 1 % non-fat dried milk with the Abcam secondary antibodies (dilution 1:10 000) anti-rabbit ab97069 and anti-mouse ab98808, protein was revealed using the chemiluminescence method according to the manufacturer's instructions (ECL SuperSignal® West Pico Chemiluminescent Substrate; Thermo Scientific). Band intensities were evaluated using Scion Image Software (Scion Corporation).
Ecl supersignal west pico chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
ECL SuperSignal® West Pico Chemiluminescent Substrate is a laboratory reagent used for the detection of proteins in Western blotting applications. It generates a luminescent signal that can be captured and quantified using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl 1d version 8, by GE Healthcare (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Scion image software, by Techcomp Instruments (1 mentions)
Affinipure goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Genegnome hr image capture system, by Syngene (1 mentions)
6 protocols using ecl supersignal west pico chemiluminescent substrate
1
Western Blot Analysis of Epididymal Adipose Tissue
2
Western Blot Analysis of PPAR-γ
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Adipose tissue samples were homogenized in RIPA buffer with a protease and phosphatase cocktail inhibitor. After determination of protein concentration by the Bradford method (Bradford, 1976 (link)), samples were diluted in Laemmli buffer and loaded (25 μg of protein) into a 12% SDS–polyacrylamide gel. Transfer to a nitrocellulose membrane was carried out using Trans-Blot Turbo-Transfer System (BioRad). Incubation with the primary antibodies was performed overnight at 4 °C in Tris-buffered saline solution containing Tween 20 (TBS-T) and 3% bovine serum albumin. Antibody dilutions were 1:1000 for PPAR-γ (Santa Cruz Biotechnology E−8 sc- 7273) and 1:1000 for beta-actin (ABCAM ab8227). After incubation overnight at 4 °C in TBS-T containing 1% nonfat dried milk with the Abcam secondary antibodies (dilution 1:1000 for anti-mouse and for anti-rabbit). Protein was revealed using the chemiluminescence method according to the manufacturer's instructions (ECL SuperSignal® West Pico Chemiluminescent Substrate, Thermo Scientific). Band intensities were evaluated using ImageQuant TL 1D Version 8.1 (GE Healthcare Life Sciences).
3
Western Blot Analysis of Adiponectin Receptors
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Renal samples were homogenized in RIPA buffer with a protease and phosphatase cocktail inhibitor. After determination of protein concentration by the Bradford method [23 (link)], samples were diluted in Laemmli buffer and loaded (50 μg of protein) into a 10% SDS–polyacrylamide gel. Transfer to a nitrocellulose membrane was carried out using Trans-Blot Turbo-Transfer System (BioRad). Incubation with the primary antibodies was performed overnight at 4°C in Tris-buffered saline solution containing Tween 20 (TBS-T) and 3% bovine serum albumin. Antibody dilutions were 1 : 1000 for Adipo-R1 (ABCAM ab126611), 1 : 1000 for Adipo-R2 (ABCAM ab77612), 1 : 500 for PPAR-α (ABCAM ab8934), 1 : 1000 for total AMPK (Cell Signaling #2532), 1 : 1000 for phospho-AMPH (Thr172) (Cell Signaling #2531), and 1 : 1000 for beta-actin (ABCAM ab8227). After incubation overnight at 4°C in TBS-T containing 1% nonfat dried milk with the Abcam secondary antibodies (dilution 1 : 3000 for anti-goat and 1 : 1000 for anti-rabbit). Protein was revealed using the chemiluminescence method according to the manufacturer's instructions (ECL SuperSignal® West Pico Chemiluminescent Substrate, Thermo Scientific). Band intensities were evaluated using ImageQuant TL 1D Version 8.1 (GE Healthcare Life Sciences).
4
Quantitative CAPN3 Protein Analysis
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The amount of CAPN3 was evaluated by Western blot (WB) analysis of muscle tissue and fibroblasts from both patients and controls. The tissues were lysed on ice with Tissue Protein Extraction Reagent T-PER™ (Thermo Scientific) and Complete Mini Anti-protease Cocktail Tablets (Roche Applied Science, Laval, PQ, Canada) according to the manufacturer’s instructions. Then, 90 μg protein for muscle samples and 40 μg protein for fibroblasts samples were separated by SDS-polyacrylamide gel electrophoresis; afterwards, we transferred electrophoretically the proteins to nitrocellulose membranes (Biorad, Hercules, CA, USA). The membranes were incubated with 1:25 rabbit anti-CAPN3 primary antibody 12A2, (Leica Biosystems, Nussloch, Germany), and 1:15000 peroxidase AffiniPure goat anti-rabbit IgG was used as a secondary antibody (111-035-045, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). GAPDH was used as an internal reference to normalize protein expression. Proteins were detected by ECL SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amount of CAPN3 and GAPDH was analyzed by densitometry using ImageJ software (https://rsb.info.nih.gov/ij , accessed on 10 December 2023).
5
Western Blot Protein Detection Protocol
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Proteins were loaded and separated on 4–12% Bis-Tris gel in MES buffer (Thermo Fisher) and transferred onto a PVDF membrane in Tris Glycine 10% ethanol for one hour at 100 V. Membranes were then treated with a blocking solution (PBS 0.1% Tween 20, 5% skimmed milk) before incubation with primary antibody (1/5000 α-GFP (Genentech, San Francisco, CA, USA), 1/1000 α-CYCLON (Bethyl laboratories, Montgomery, TX, USA) in PBS, 0.1% Tween 20, 5% skimmed milk), PBS 0.1% Tween washes and secondary antibody incubation (1/5000 anti-rabbit HRP (BioRad, Marne-La-Coquette, France). Revelation was performed through incubation in ECL SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and images captured using Vilber imaging system. The original blots are shown in the Supplementary Materials .
6
Protein Expression Analysis in HUVECs
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Total proteins of HUVECs in 6-well plates were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (20 uL/mL) and phosphatase inhibitors (20 uL/mL). Then proteins (20ul samples per lane) were separated on 10% polyacrylamide gels by SDS-PAGE and transferred onto PVDF membranes. After blocked with 5% skimmed milk for 1 h.
At room temperature, the blots were incubated with various concentrations of primary antibodies at 4 °C overnight according to the manufacturer’s protocols. Then the membranes were washed three times in TBS-T for 5 min each time and subsequently incubated with horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit antibodies (1:10,000 dilution) for 2 h, followed by three washes with TBS-T 5 to 7 min each time. Lastly, the blots incubated with ECL Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) and detected using the GeneGnome HR Image Capture System (Syngene).
At room temperature, the blots were incubated with various concentrations of primary antibodies at 4 °C overnight according to the manufacturer’s protocols. Then the membranes were washed three times in TBS-T for 5 min each time and subsequently incubated with horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit antibodies (1:10,000 dilution) for 2 h, followed by three washes with TBS-T 5 to 7 min each time. Lastly, the blots incubated with ECL Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) and detected using the GeneGnome HR Image Capture System (Syngene).
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