BHK-21 cells were transfected with vector (pcDNA3.1) alone or pcDNA3.1-HA-M and pcDNA3.1 or pcDNA3.1-HA-M and pcDNA3.1-AnxA2-Flag, and at 24 hpt, transfected cells were collected and washed with PBS. Samples were harvested via low-temperature low-speed centrifugation and were cleaned by using cold PBS. The cytoplasmic and plasma membrane (PM) protein components were separated and collected by using the MinuteTM Plasma Membrane Protein Isolation and Cell Fractionation kit (Invent Biotechnologies, USA) [39 (link)]. The amounts of protein in the cytoplasm and the PM were determined by western blot analysis with the addition of β-actin (Abways) and Na/K ATPase (Abways) antibodies.
Protocol detail
Membrane Protein Isolation and Fractionation
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Actin
Antibodies
Anxa2
Cell fractionation
Cells
Centrifugation
Cold
Cytoplasm
Isolation
Membrane
Membrane protein
Na k atpase
Plasma
Plasma membrane
Plasma protein
Protein
Vector
Western blot analysis
Corresponding Organization :
Other organizations : Shandong Normal University
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Variable analysis
independent variables
- Transfection with vector (pcDNA3.1) alone
- Transfection with pcDNA3.1-HA-M and pcDNA3.1
- Transfection with pcDNA3.1-HA-M and pcDNA3.1-AnxA2-Flag
- Amounts of protein in the cytoplasm and the plasma membrane (PM) measured by western blot analysis
- BHK-21 cells
- Collection and washing of transfected cells with PBS
- Harvesting of samples via low-temperature low-speed centrifugation
- Cleaning of samples using cold PBS
- Separation and collection of cytoplasmic and plasma membrane (PM) protein components using the MinuteTM Plasma Membrane Protein Isolation and Cell Fractionation kit
- Addition of β-actin (Abways) and Na/K ATPase (Abways) antibodies for western blot analysis
- Positive control: Not specified
- Negative control: Transfection with vector (pcDNA3.1) alone
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