After cells were lysed with ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Changzhou, China) for 30 min on ice, lysates were centrifuged at 13,000 ×g with 20 min and the supernatants were used as total cell lysates. Protein concentration was determined by Bradford protein assay (Bio-Rad, USA). A quantity of 50 μg total protein per lane was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Amersham Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% milk powder in 0.05% Tween-TBS, incubated with the specific antibodies, including Cyclin E1, p27, and CDK7 and CDC7 rabbit monoclonal antibodies (Epitomics, Burlingame, CA, USA). Detection of the target proteins on the membranes was performed using the ECL western blotting detection reagents. Signal density was detected using the MF-ChemiBIS Family including gel capture software (DNR Bio-Image system, Ltd., Jerusalem, Israel) and quantitatively analyzed by densitometry using the Image J software.
Amersham hybond p polyvinylidene fluoride pvdf membranes
Manufactured by GE Healthcare
Sourced in United Kingdom
Amersham Hybond-P is a polyvinylidene fluoride (PVDF) membrane designed for use in Western blotting applications. PVDF membranes are commonly used for the immobilization and detection of proteins that have been separated by gel electrophoresis.
Automatically generated - may contain errors
2 protocols using amersham hybond p polyvinylidene fluoride pvdf membranes
1
Cell Lysis and Western Blot Analysis
2
Western Blot Analysis of Clioquinol Protein
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The cells were incubated with 5 mmol L -1 clioquinol or 0.1 % DMSO for 72 h. Cell lysates were obtained using standard techniques and RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Protein concentration was determined by the Bradford protein assay (Bio-Rad, uSA). A quantity of 50 μg total protein per lane was separated by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Amersham Hybond-P polyvinylidene fluoride (PVDf) membranes (GE Healthcare). Membranes were blocked with 5 % milk powder in 0.05 % Tween-Tris buffer saline (TBS), incubated with specific primary antibodies and then with the appropriate horseradish peroxidase-conjugated secondary antibodies (Sinbio, China). GAPDH was used as an internal standard. Detection of the target proteins on the membranes was performed using the ECL western blotting detection reagents (GE Healthcare, uK). Signal density was detected using the Mf-ChemiBIS family including gel capture software (DNR Bio-Image system, Ltd., Israel) and quantitatively analyzed by densitometry using the Image J software.
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