Cultured cells were lysed for 30 min in ice-cold RIPA lysis buffer (Servicebio, Wuhan, China) containing protease inhibitors (Servicebio, Wuhan, China). Protein concentration was measured by the Coomassie brilliant blue G-250 (BioFroxx; neoFroxx GmbH) staining method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for protein electrophoresis. The protein was transferred to a polyvinylidene difluoride membrane. The PVDF membrane was blocked in 5% BSA for 1h and then incubated with diluted primary antibodies for 12 h at 4 °C. After washing with TBST three times, the PDVF membrane was incubated in diluted HRP Goat Anti-Rabbit IgG (1:5000, A21020, abbkine) or HRP Goat Anti-mouse IgG (1:5000, A21010, abbkine) at room temperature for 1h. The proteins were detected by the ECL system (Bio-Rad) and analyzed by Image Lab (6.0.1). All primary antibodies were shown as follows: GAPDH (1:2500, rabbit, YM1235, Immunoway), CDC42 (1:200, mouse, sc-8401, SANTA CRUZ BIOTECHNOLOGY).
Coomassie brilliant blue g 250
Manufactured by neoFroxx
Sourced in China
Coomassie brilliant blue G-250 is a dye commonly used in protein quantification and detection methods in laboratory settings. It is a blue, crystalline powder that forms a blue-colored complex when bound to proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Wuhan Servicebio Technology (1 mentions)
A21010, by Abbkine (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Sc 8401, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
A21020, by Abbkine (1 mentions)
Gelatin, by Maclin Biochemical Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ant020, by Antgene (1 mentions)
3 protocols using coomassie brilliant blue g 250
1
Protein Extraction and Western Blot Analysis
2
Gelatin Zymography for MMP Activity
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Total proteins were extracted from culture supernatants and separated via PAGE on 7.5% polyacrylamide gels containing 0.5% gelatin (Macklin, Shanghai, China). Gels were then equilibrated with 2.5% Triton X-100 and incubated in substrate buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 10 mmol/L CaCl2) for 24 h at 37 °C. Gels were stained with Coomassie Brilliant Blue G250 (BioFROXX, Guangzhou, China) for 2 h and washed until clear zones associated with MMP activity were observed.
3
Western Blot Analysis of BDKRB2, VEGF
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The cells were harvested and lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) supplemented with a protease inhibitor cocktail (Roche). The protein concentration was measured using the Coomassie brilliant blue G-250 (Biofroxx) staining method. Protein samples (40 μg each) were separated by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane and immunoblotted with primary antibodies. The primary antibodies used were as follows: Rabbit monoclonal anti-human BDKRB2 (1:500; ab134118, Abcam), rabbit polyclonal anti-human VEGF (1:1,000; 102643, Genetex), rabbit polyclonal anti-human α-tubulin (1:2,000; ANT014, Antgene). The primary antibodies were incubated overnight at 4°C. The membranes were then washed with phosphate-buffered saline Tween-20 (PBST, pH 7.5) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,000; ANT020, Antgene) for 1 h at 37°C. Finally, the proteins were detected using an enhanced chemiluminescence system (Pierce/Thermo Fisher Scientific).
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