Cells were lysed in TritonX-100 with protease and phosphatase inhibitor cocktail (Boston Bioproducts). Cell lysates were separated by electrophoresis on 8% SDS- polyacrylamide gels and transferred to Immobilon ® polyvinylidene difluoride membrane. The membrane was blocked in 5% milk in TBS with 0.1% Tween-20 (TBS-Tw) for 1 h. The blot was then incubated with the following primary antibodies anti-TRPV4 (1:300) (Alomone), anti-Actin (1:1000). The ECL (Pierce West Pico) method was used with anti-rabbit (Jackson Laboratories) at a dilution of 1:10,000 and developed using Kodak X-ray film or Protein Simple. Results were quantified using Image J software.
Protease and phosphatase inhibitor cocktail
Manufactured by Boston BioProducts
The Protease and Phosphatase Inhibitor Cocktail is a ready-to-use solution designed to inhibit a broad range of proteases and phosphatases. It is a versatile tool for preserving the integrity of proteins and enzymes during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using protease and phosphatase inhibitor cocktail
1
Western Blot Protein Detection Protocol
2
Western Blot Protein Detection Protocol
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Cells were lysed in TritonX-100 with protease and phosphatase inhibitor cocktail (Boston Bioproducts). Cell lysates were separated by electrophoresis on 8% SDS- polyacrylamide gels and transferred to Immobilon ® polyvinylidene difluoride membrane. The membrane was blocked in 5% milk in TBS with 0.1% Tween-20 (TBS-Tw) for 1 h. The blot was then incubated with the following primary antibodies anti-TRPV4 (1:300) (Alomone), anti-Actin (1:1000). The ECL (Pierce West Pico) method was used with anti-rabbit (Jackson Laboratories) at a dilution of 1:10,000 and developed using Kodak X-ray film or Protein Simple. Results were quantified using Image J software.
3
Western Blot Protein Detection Protocol
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Cells or tumor tissues were lysed using RIPA lysis buffer (Cat. No. BP-115DG, Boston Bio Products, Ashland, MA) supplemented with protease and phosphatase inhibitor cocktail (Cat. No. PPC1010, Sigma-Aldrich, St. Louis, MO) as described previously.(26 (link), 32 (link)) Briefly, an equal amount of protein samples (20–40 μg/lane) were loaded to precast gels and transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) non-fat dry milk in TBST buffer, and subsequently probed with primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibody for 1–2 h at room temperature. Finally, the membranes were incubated with chemiluminescent HRP substrate (Cat. No. WBKLS0500, Millipore Sigma, Billerica, MA), and were recorded using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). The immunoblots were quantified using Image J software. The primary antibody details are provided in Supplementary Table 1 .
4
Western Blot Protein Detection Protocol
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Cells or tumor tissues were lysed using RIPA lysis buffer (Cat. No. BP-115DG, Boston Bio Products, Ashland, MA) supplemented with protease and phosphatase inhibitor cocktail (Cat. No. PPC1010, Sigma-Aldrich, St. Louis, MO) as described previously.(26 (link), 32 (link)) Briefly, an equal amount of protein samples (20–40 μg/lane) were loaded to precast gels and transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) non-fat dry milk in TBST buffer, and subsequently probed with primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibody for 1–2 h at room temperature. Finally, the membranes were incubated with chemiluminescent HRP substrate (Cat. No. WBKLS0500, Millipore Sigma, Billerica, MA), and were recorded using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). The immunoblots were quantified using Image J software. The primary antibody details are provided in Supplementary Table 1 .
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