Related cell lysates were run on 4–12% SDS PAGEs to conduct Western blot analyses. RIPA lysis buffer containing protease inhibitors (#KGP250, KeyGEN BioTECH, Nanjing, China) was used to extracted PCa cells protein following to the operation protocol. Then, equal amounts of 30 μg proteins were separated by SDS/PAGEs and transferred onto PVDF membranes (Millipore, Billerica, MA, USA) electrically. Then, membranes were blocked with TBS(Tris/saline solution with 0.1% Tween-20) including 5% milk without fat for 1 h and incubated overnight at 4 °C covering by unique antibodies: rabbit anti-β-actin (#4970, CST), rabbit anti-Slug (#9585), rabbit anti-ZEB1(#3396, CST), rabbit anti-E-Cadherin (#3195, CST) and rabbit anti-hnRNPL (4D11, #ab6106, Abcam). All membranes were subsequently incubated at room temperature for 1 h with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse IgG antibodies (Cell Signaling Technology). Final bands were visualized by ECL kit (Pierce Biotechnology, Rockford, IL, USA). Image J was applied to quantify the intensity of the band. Experiments were performed in triplicate.
Horseradish peroxidase linked secondary anti rabbit or anti mouse igg antibodies
Manufactured by Cell Signaling Technology
Horseradish peroxidase-linked secondary anti-rabbit or anti-mouse IgG antibodies are laboratory reagents used to detect the presence of primary antibodies raised against rabbit or mouse immunoglobulin G (IgG) in immunoassays. The horseradish peroxidase enzyme linked to the secondary antibody generates a colorimetric or chemiluminescent signal upon addition of a suitable substrate, allowing for the visualization and quantification of the target protein.
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2 protocols using horseradish peroxidase linked secondary anti rabbit or anti mouse igg antibodies
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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Related cell lysates were run on 4-12% SDS PAGEs to conduct Western blot analyses. RIPA lysis buffer containing protease inhibitors (#KGP250, KeyGEN BioTECH, Nanjing, China) was used to extracted PCa cells protein following to the operation protocol. Then, equal amounts of 30ug proteins were separated by SDS/PAGEs and transferred onto PVDF membranes (Millipore, Billerica, MA, USA) electrically. Then, membranes were blocked with TBS(Tris/saline solution with 0.1% Tween-20) including 5% milk without fat for 1h and incubated overnight at 4 ℃ covering by unique antibodies: rabbit anti-β-actin (#4970, CST), rabbit anti-Slug (#9585), rabbit anti-ZEB1(#3396, CST), rabbit anti-E-Cadherin (#3195, CST) and rabbit anti-hnRNPL (4D11, #ab6106, Abcam). All membranes were subsequently incubated at room temperature for 1 hour with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse IgG antibodies (Cell Signaling Technology). Final bands were visualized by ECL kit ((Pierce Biotechnology, Rockford, IL, USA). Image J was applied to quantify the intensity of the band
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