Immunoblotting and Immunoprecipitation of Kidney BBMVs

BBMVs prepared using the Ca²⁺ precipitation method, cortical membrane, and whole homogenate were obtained from mouse kidneys and used for immunoblotting and immunoprecipitation analyses as described previously^{35 (link),45 (link),48 (link),49 (link)}. Protein samples were heated at 95 °C for 5 min in sample buffer in the presence of 2-mercaptoethanol and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA, USA) and treated with diluted antibodies. Signals were detected using Immobilon Western (Millipore)^{35 (link),45 (link),48 (link),49 (link)}.

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Sasaki S., Shiozaki Y., Hanazaki A., Koike M., Tanifuji K., Uga M., Kawahara K., Kaneko I., Kawamoto Y., Wiriyasermkul P., Hasegawa T., Amizuka N., Miyamoto K.I., Nagamori S., Kanai Y, & Segawa H. (2022). Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia. Scientific Reports, 12, 6353.

Publication 2022

Immobilon-P polyvinylidene difluoride Immobilon Western

Antibodies Buffer Cortical Electrophoresis Immobilon Immobilon p Immunoprecipitation Kidneys Membrane Mercaptoethanol Mouse Polyvinylidene difluoride Proteins Sds page

Corresponding Organization :

Other organizations : Tokushima University, Osaka University, Jikei University School of Medicine, Hokkaido University, Ryukoku University

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Variable analysis

independent variables

Ca2+ precipitation method
Cortical membrane
Whole homogenate

dependent variables

Immunoblotting
Immunoprecipitation

control variables

Protein samples were heated at 95 °C for 5 min in sample buffer in the presence of 2-mercaptoethanol
Separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA, USA) and treated with diluted antibodies
Signals were detected using Immobilon Western (Millipore)

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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