Neurons were lysed in 1×RIPA buffer (Pierce Biotechnology, Rockford, IL) with complete phosphatase and protease inhibitors (Roche, Indianapolis, IN) and spun at 1,000×g for 10 min to obtain supernatants. Protein concentrations were measured using BCA protein assay kit (Pierce). For immunoprecipitations, supernatants (300 µg) were first precleared with protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and mixed with anti-STEP antibody (4 µg) overnight at 4°C as described [37 (link)]. On the second day, protein A/G-agarose was added to the antibody-bound complex for 4 h at 4°C and precipitates were washed 3 times with 1×RIPA buffer and analyzed by SDS-PAGE and immunoblotting.
Protocol detail
Immunoprecipitation and Western Blot Analysis
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Agarose
Anti antibody
Antibody
Assay
Buffer
G protein
Immunoprecipitations
Neurons
Phosphatase
Protease inhibitors
Protein
Protein a
Protein g
Ripa
Sds page
Corresponding Organization :
Other organizations : Yale University
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Variable analysis
independent variables
- Lysis of neurons in 1xRIPA buffer with phosphatase and protease inhibitors
- Centrifugation at 1,000xg for 10 minutes to obtain supernatants
- Immunoprecipitation of supernatants (300 µg) with anti-STEP antibody (4 µg) overnight at 4°C
- Addition of protein A/G-agarose beads to the antibody-bound complex for 4 hours at 4°C
- Protein concentrations measured using BCA protein assay kit
- Analysis of precipitates by SDS-PAGE and immunoblotting
- Incubation time and temperature for immunoprecipitation (overnight at 4°C)
- Incubation time and temperature for binding of protein A/G-agarose beads (4 hours at 4°C)
- Washing of precipitates with 1xRIPA buffer 3 times
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