Renal extracts from whole-kidney homogenates and NRK cells were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1mM phenylmethylsulfonyl fluoride (PMSF), 1.2 mM Na3VO4, 2.5 mM NaF, and 1 mM DTT (Sigma-Aldrich, USA) and protease inhibitor cocktail (Pierce, USA). 11 (link) After lysis, the extracts were centrifuged (16000 g for 20 min at 4 °C), and the supernatant was saved as the NRK cell extract. Protein concentrations were determined with the BCA Protein Assay kit (Pierce, USA). Renal extracts (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with antibodies to β5 subunit (1:1000; Abcam, #ab3330), α3 subunit (1:1000; Abcam, #ab119419), or β-actin (loading control, 1:1000; Sigma-Aldrich, #A5441). Probed membranes were washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000; Seracare KPL), and assayed for enhanced chemiluminescence (Thermo Fisher Scientific, USA). Densitometry was performed with AlphaEase FC software (Alpha Innotech, USA).
Ab119419
Manufactured by Merck Group
Ab119419 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, but its core function is not specified in the information provided. A more detailed and unbiased description cannot be given without potentially extrapolating or interpreting beyond the available facts.
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Lab products found in correlation
2 protocols using ab119419
1
Renal Protein Extraction and Western Blotting
2
Renal Protein Extraction and Western Blotting
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Renal extracts from whole-kidney homogenates and NRK cells were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1mM phenylmethylsulfonyl fluoride (PMSF), 1.2 mM Na3VO4, 2.5 mM NaF, and 1 mM DTT (Sigma-Aldrich, USA) and protease inhibitor cocktail (Pierce, USA). 11 (link) After lysis, the extracts were centrifuged (16000 g for 20 min at 4 °C), and the supernatant was saved as the NRK cell extract. Protein concentrations were determined with the BCA Protein Assay kit (Pierce, USA). Renal extracts (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with antibodies to β5 subunit (1:1000; Abcam, #ab3330), α3 subunit (1:1000; Abcam, #ab119419), or β-actin (loading control, 1:1000; Sigma-Aldrich, #A5441). Probed membranes were washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000; Seracare KPL), and assayed for enhanced chemiluminescence (Thermo Fisher Scientific, USA). Densitometry was performed with AlphaEase FC software (Alpha Innotech, USA).
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