To quantify HC1 and HC2 protein expression, first instar larvae dissected from the uteri were homogenized twice in ice-cold lysis buffer which contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Na-deoxycholate, 0.5% NP-40, 0.5% SDS, and protease inhibitor (EDTA-free). Lysates were cleared by centrifugation at 15.000×g at 4 °C for 30 min, and the protein content was determined by a protein assay using bovine serum albumin (BSA) as a standard (Bradford method). Sixty micrograms of protein from each sample was reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20 and then incubated overnight at 4 °C with anti-HC1 or HC2 antibodies diluted 1:500. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz, USA) diluted 1:1000. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (Advansta Inc., Menlo Park, USA) and visualized using the Chemidoc™ XRS + System (Bio-Rad, Hercules, USA).
Mini protean tgx system precast protein gels
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protean TGX System Precast Protein Gels are a lab equipment product designed for electrophoresis separation and analysis of proteins. The gels are pre-cast and ready-to-use, providing a convenient and consistent solution for protein separation and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (4 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Trans blot turbo transfer system apparatus, by Bio-Rad (1 mentions)
Erα sc 8002, by Santa Cruz Biotechnology (1 mentions)
Antivaspin antibody, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Chemidoctm xrs system, by Bio-Rad (1 mentions)
4 protocols using mini protean tgx system precast protein gels
1
Quantification of HC1 and HC2 Proteins
2
Western Blot Analysis of Apelin, Cyclins, and Kinases
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The western blotting procedure used to determine apelin/APJ, cyclins and kinases proteins expression was described previously (33 (link)). Briefly, the proteins (80 µg) were separated by 4-20% Mini-Protean TGX System Precast Protein Gels and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad Laboratories, Inc.). The membranes were blocked for 1 h in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween-20 at room temperature (RT), then incubated overnight at 4°C with appropriate primary antibodies (Table I ). The membranes were then washed in Tris-buffered saline containing 0.1% Tween-20 and incubated for 1 h at RT with a horseradish peroxidase-conjugated secondary antibody (Table I ). β-actin was used as a loading control. Signals were detected by chemiluminescence using a WesternBright™Sirius (cat. no. K-12043-D20; Advansta, Inc.) and visualised using the Chemidoc™ XRS+ System (Bio-Rad Laboratories, Inc.). All bands were quantified using a densitometer and ImageJ software (version 1.51; US National Institutes of Health).
3
Western Blot Analysis of Apoptosis Markers
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After treating cells with test compounds for 24 or 48 h, cells were lysed in lysis buffer. Proteins were separated in 4-20% Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad) using a Trans-Blot Turbo Transfer System apparatus (Bio-Rad). The blots were blocked for 1 h in 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 and then incubated overnight at 4 °C with antibodies specific for caspase-3 and cleaved caspase-3 (#9662, Cell Signaling Technology), PARP (#9542, Cell Signaling Technology), ERα (sc-8002, Santa Cruz Biotechnology), ERβ (sc-8974, Santa Cruz Biotechnology), and GPR30 (ab3974, Abcam). The membranes were then washed three times in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated antirabbit (#7074) secondary antibodies (Cell Signaling Technology). β-Actin (Cat. No. A5316, Sigma-Aldrich) was used as a loading control. Immunopositive bands were visualized using WesternBright Quantum HRP substrate (Cat. No. K-12043 D20, Advansta Inc., Menlo Park, CA, USA). Quantification of protein bands was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, LLC, Upland, CA, USA).
4
Western Blotting of Vaspin Protein
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Tissue preparation, lysis, Western blotting and quantification were performed as previously described (Rak et al. 2015a,b) . For each sample, 30 μg of protein were reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad), and then transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20, and then incubated overnight at 4°C with antivaspin antibody (cat no. PA5-30989, ThermoFisher Scientific) diluted at 1:1,000. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated antibody (cat. no. 7074, Cell Signaling Technology) diluted at 1:1000. An anti-β-actin antibody (cat no. A5316, Sigma-Aldrich) was used as loading control. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (cat. no. K-12043 D20, Advansta Inc., Menlo Park, USA) and visualised using the Chemidoc TM XRS + System (Bio-Rad). All visible bands were quantified using a densitometer and ImageJ software (US National Institutes of Health).
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