Whole-cell lysates were prepared in RIPA buffer [10 mM Tris–HCl (pH 8.0), 140 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA, 0.1% SDS (wt/vol), 0.1% sodium deoxycholate (wt/vol) and 1% Triton X-100 (vol/vol)] containing protease inhibitors (Sigma-Aldrich). Protein extracts were run on any kD Mini-PROTEAN TGX precast gel (Bio-Rad) and blotted on nitrocellulose membrane. Blots were blocked in 5% non-fat milk dissolved in 1x PBS, 0.1% Tween-20 (blocking solution). After the incubation with the specific primary antibody, secondary antibody incubation was carried out for 1 h (1:3000 in blocking solution) at RT. Blots were developed with SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific). The chemiluminescent signals were detected with a ChemiDoc Imaging system (BioRad).
Kd mini protean tgx precast gel
Manufactured by Bio-Rad
Sourced in United States
The KD Mini-PROTEAN TGX precast gel is a ready-to-use polyacrylamide gel designed for electrophoretic separation of proteins. It is compatible with the Mini-PROTEAN electrophoresis system.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mini protean electrophoresis cell, by Bio-Rad (1 mentions)
Ez run prestained rec protein ladder, by Thermo Fisher Scientific (1 mentions)
3 protocols using kd mini protean tgx precast gel
1
Western Blot Protocol for Protein Detection
2
Western Blot Analysis of Exosomal Markers
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15 μg of protein was heated at 95°C for 3 min, loaded onto any kD Mini-PROTEAN TGX Precast Gel (Bio-Rad, Hercules, CA, USA), and transferred to polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Blots were blocked with Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% nonfat dry milk for 1 hour at room temperature (RT). Membranes were then incubated overnight at 4°C with the following antibodies: exosomal marker antibodies CD9, CD63, CD81, HSP70 (diluted 1 : 1000) from the ExoAB-KIT-1 Western blot antibody detection kit (System Biosciences), anti-CD39 (clone BU61, diluted 1 : 500), and anti-CD73 (clone 4G4 from Abcam, diluted 1 : 50). Blots were then washed with TBS-T and incubated with the appropriate HRP-conjugated secondary antibody (System Biosciences, dilution 1 : 20,000) for 1 hour at RT. Membranes were then incubated with Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA, USA) for 1 min and exposed on X-ray films.
3
SDS-PAGE Analysis of Skin Tissue Proteins
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1D SDS-PAGE was performed to observe protein bands in the skin tissue sample. 100 mg of each sample were homogenized with 300 µl saline and were then subjected to cooling centrifugation (VS-18000 M, Korea, power 220 V/50 Hz) (12,303×g for 30 min at 4 °C). 100 µl from the supernatant proteins were mixed with 500 µl acetone and left overnight at -20 °C. Before measurement, samples were centrifuged at 12,303×g for 3 min at 4 °C. Proteins were diluted with sample buffer (10% SDS, 20% Glycerol, 0.2 M trispH6.8, 10 mM beta-mercapto-ethanol, and 0.05% bromophenol blue) at the ratio of 1:4 and were then heated for 5 min at 95 °C. Proteins were loaded into the well of kD™ Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad, Hercules, CA), and were run with Mini-PROTEAN electrophoresis cells (Bio-Rad, Hercules, CA) at 80 V for 4 h. EZ-Run Prestained Rec Protein Ladder (Fisher Bioreagents, Pittsburgh, PA) containing proteins from 16 to 270 kDa was used as a protein marker. Gels were stained with staining solution (50% dH 2 O, 40% methanol, and 10% glacial acetic acid) [43] .
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