Synaptoneurosomes were isolated from mouse cortex (P28) as described [28 (link)]. Tissue was homogenized in 10 mM HEPES, 1 mM EDTA, 2 mM EGTA, 200 μM Na3VO4, 10 mM NaF, and protease inhibitors. After sonication, the sample was filtered through a 100 μm filter and 5 μm cell strainers, then centrifuged at 1000 × g for 10 min. The pellet was resuspended in 500 μl of RIPA buffer (20 mM Tris pH 7.0, 0.15 M NaCl, 5 mM EDTA, 1 mM EGTA, 1% NP-40, 1% deoxycholate, 0.1% SDS, 200 μM Na3VO4, 10 mM NaF, protease inhibitors), agitated for 40 min, and re-centrifuged at 16,000 × g for 10 min. The supernatant was collected as the synaptoneurosome fraction. Precleared lysates (0.5–1 mg) were incubated with mouse monoclonal anti-TIAM1 (Santa Cruz sc-393315) or normal IgG for 2 hr at 4°C and collected on Protein A/G Sepharose beads. SDS-PAGE and immunoblotting with rabbit NrCAM antibodies was carried out, then filters were stripped and reprobed for Tiam1/2.
Sc 393315
Manufactured by Santa Cruz Biotechnology
Sc-393315 is a lab equipment product offered by Santa Cruz Biotechnology. It is a multi-purpose device designed for various laboratory applications. The core function of Sc-393315 is to facilitate [CORE_FUNCTION]. No further details are available without the risk of extrapolation or bias.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sc 13152, by Santa Cruz Biotechnology (1 mentions)
Tiam1, by Santa Cruz Biotechnology (1 mentions)
Sc 514583, by Santa Cruz Biotechnology (1 mentions)
Ab254349, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Synapsin, by Abcam (1 mentions)
Psd 95, by Santa Cruz Biotechnology (1 mentions)
Glur1, by Santa Cruz Biotechnology (1 mentions)
Sc 32290, by Santa Cruz Biotechnology (1 mentions)
3 protocols using sc 393315
1
Isolating Synaptoneurosomes from Mouse Cortex
2
Hippocampal Protein Extraction and Analysis
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Each gram of hippocampi were homogenized with 30 µl cocktail protease inhibitor (Roche Molecular Biochemicals, Germany) and 3 ml RIPA buffer (US Biological, USA). After centrifugation at 12000 rpm (4 •C), the supernatant was collected and stored at -20 •C. BCA assay and spectrophotometry were used to assess the total protein concentration. The supernatant sample (50µg of protein) was separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore Inc., Darmstadt, Germany). After blocking with 5% skim milk for 2 hours, the membrane was incubated with primary antibody overnight: Rac1 (1:500, sc-514583, Santa Cruz Biotechnology), Tiam1 (1:500, sc-393315, Santa Cruz Biotechnology), α-chimaerin (1:500, sc-365985, Santa Cruz Biotechnology), Bcr (1:500, sc-104, Santa Cruz Biotechnology), GluR-1 (1:500, sc-13152, Santa Cruz Biotechnology), Synapsin 1(1:500, ab254349, Abcam), and PSD-95 (1:500, sc-32290, Santa Cruz Biotechnology). The speci city of these primary antibodies has been veri ed (Mao et Tsai et al. 2012 (link)). The membrane was incubated with a mixture of anti-rabbit IgG (Golden Bridge, Zhongshan, China) and horseradish peroxidase (HRP) for 1 hour at room temperature. Quantity One software (Bio-Rad, USA) was used for quantitative analysis.
3
Isolation and Characterization of Synaptoneurosomes
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Synaptoneurosomes were isolated from mouse cortex (P28) as described [28] (link). Tissue was homogenized in 10 mM HEPES, 1 mM EDTA, 2 mM EGTA, 200 µM Na3VO4, 10 mM NaF, and protease inhibitors. After sonication, the sample was filtered through a 100 μm filter and 5 μm cell strainers, then centrifuged at 1000 × g for 10 min.
The pellet was resuspended in 500 μl of RIPA buffer (20 mM Tris pH 7.0, 0. 10 mM NaF, protease inhibitors), agitated for 40 min, and re-centrifuged at 16,000 × g for 10 min. The supernatant was collected as the synaptoneurosome fraction.
Precleared lysates (0.5-1 mg) were incubated with mouse monoclonal anti-TIAM1 (Santa Cruz sc-393315) or normal IgG for 2 hr at 4°C and collected on Protein A/G Sepharose beads. SDS-PAGE and immunoblotting with rabbit NrCAM antibodies was carried out, then filters were stripped and reprobed for Tiam1/2.
The pellet was resuspended in 500 μl of RIPA buffer (20 mM Tris pH 7.0, 0. 10 mM NaF, protease inhibitors), agitated for 40 min, and re-centrifuged at 16,000 × g for 10 min. The supernatant was collected as the synaptoneurosome fraction.
Precleared lysates (0.5-1 mg) were incubated with mouse monoclonal anti-TIAM1 (Santa Cruz sc-393315) or normal IgG for 2 hr at 4°C and collected on Protein A/G Sepharose beads. SDS-PAGE and immunoblotting with rabbit NrCAM antibodies was carried out, then filters were stripped and reprobed for Tiam1/2.
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