Sperm cells were solubilized using commercially available RIPA cell lysis buffer (AMRESCO, Ohio, USA) in combination with sonication. Equivalent amount of total protein extract was resuspended with an appropriate volume of Lithium dodecyl sulfate (LDS) loading buffer (Invitrogen) in the presence of reducing agent (50 mM DTT). Samples were heated in a 100 °C dry bath for 10 min and cooled on ice before loading on gels. Proteins were separated by SDS-PAGE (gradient T-Pro EZ Gel Solution, T-Pro Biotechnology, NTC, TW) and wet-blotted onto a PVDF membrane (Immobilon-P, Millipore, Billerica, MA, USA). After blocking for 1 h with blocking buffer (5 mM Tris, 250 mM sucrose, pH 7.4 with 0.05% v/v Tween-20 [TBST], supplemented with 5% milk powder) at RT, blots were incubated with anti-Phosphotyrosine (Merk/Millipore) primary antibody and subsequently with secondary antibody (1:5000 dilution in TBST) for 1 h at RT. Protein signals were visualized by chemiluminescence (Merck, Ltd., TW) and were detected with ChemiDoc™ XRS+ system (Bio-Rad).
Ripa cell lysis buffer
Manufactured by Avantor
Sourced in United States
RIPA cell lysis buffer is a commonly used buffer for the extraction and solubilization of proteins from cells. It contains a combination of detergents, salts, and other components that help to disrupt cell membranes and release the contents of the cells, including proteins. This buffer is suitable for a variety of cell types and is often used in protein analysis and purification applications.
Automatically generated - may contain errors
2 protocols using ripa cell lysis buffer
1
Phosphotyrosine Western Blot Analysis
2
Western Blot Analysis of P-gp in BCSCs
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The P-gp was selected as a representative protein for confirmation of RT-PCR gene expression analysis. For this, total protein was extracted from cells and Western blot analysis was applied for the target protein P-gp. Total lysate of silenced and MOCK control BCSC-like (5 × 10 6 ) cells were prepared in RIPA cell lysis buffer (Amresco, USA) as previously mentioned [13] . The protein concentration of the cell lysate was determined as previously described [13] . Protein lysates were analyzed on SDS-PAGE and transferred onto 0.45 µm nitrocellulose membrane for 5 h using BioRad Electro Blot System. The membrane was hybridized with antibodies (Rabbit Anti-Human GAPDH antibody, 1:200 dilution; Rabbit Anti-Human-Pgp antibody, 1:1000; ABCAM). Then membrane was hybridized with secondary antibody (HRP conjugated anti-Rabbit for 1 h). After washing, the detection buffer ECL substrate (Abcam, USA) was added the membrane was analyzed under chemiluminescent gel imaging system and density of each protein band was measured (Vilber-smart imaging, Cedex, France). The band densities of P-gp was normalized by the band densities of GAPDH for drug resistant and silenced BCSC-like cells.
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