Expression and phosphorylation of SmAkt proteins in oocytes were confirmed by immunoprecipitation of oocyte lysates according to the procedure described previously (Vicogne et al., 2004 (link)). Following 15 h of expression, oocytes were lysed in buffer A (50 mM Hepes pH 7.4, 500 mM NaCl, 0.05% SDS, 5 mM MgCl2, 1 mg/mL bovine serum albumin, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL benzamidine, 1 mM PMSF, 1 mM sodium vanadate) containing 0.5% Triton X-100 and centrifuged at 12,000g for 15 min at 4 °C. Lysates were incubated with anti-V5 antibodies (1:100, Invitrogen) for 4 h at 4 °C. Protein A-Sepharose beads (5 mg; Amersham Biosciences) were added for 1 h at 4 °C. Beads were washed three times and resuspended in Laemmli sample buffer. Eluted immune complexes were subjected to a 10% SDS–PAGE, then analyzed by Western blotting using anti-V5 (1:50,000) or anti-phospho-T308 Akt (1:5000; Upstate Biotechnology) antibodies. Mouse or rabbit Trueblot® secondary antibodies (eBioscience) were used as secondary antibodies and chemoluminescence was revealed using the advanced ECL detection system (Amersham Biosciences).
Advanced ecl detection system
Manufactured by Cytiva
The Advanced ECL detection system is a lab equipment product that provides chemiluminescent detection capabilities for Western blot analysis. It is designed to offer sensitive and quantitative detection of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protein a sepharose beads, by Cytiva (3 mentions)
Anti ha, by Thermo Fisher Scientific (2 mentions)
Anti myc, by Thermo Fisher Scientific (2 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Trueblot secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti myc, by Merck Group (1 mentions)
3 protocols using advanced ecl detection system
1
Immunoprecipitation of SmAkt Proteins
2
Immunoprecipitation of Schistosoma Proteins
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Immunoprecipitation of SmHDAC8, SmDia-RBD and SmRho1 (isoforms and mutant constructs) proteins expressed in oocytes was performed using HA and Myc tags respectively. 15h after cRNA injection in the equatorial region, oocytes were lysed in buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.05% SDS, 0.5% Triton X100, 5 mM MgCl2, 1 mg mL-1 bovine serum albumin, 10 mg mL-1 leupeptin, 10 mg mL-1 aprotinin, 10 mg mL-1 soybean trypsin inhibitor, 10 mg mL-1 benzamidine, 1 mM sodium vanadate) and centrifuged at 4°C for 15 min at 10 000 x G. Supernatants were incubated with anti-Myc (1/100; Invitrogen) and anti-HA (1/100 Invitrogen) antibodies for 4 h at 4°C. Protein A-Sepharose beads (5 mg, Amersham Biosciences) were added for 1 h at 4°C. Immune complexes were collected by centrifugation, rinsed three times, resuspended in Laemmli sample buffer, and subjected to a 10% SDS-PAGE. Immune complexes were analyzed by Western Blotting using anti-Myc (1/50 000, Sigma Aldrich) or anti-HA (1/10 000, Invitrogen) antibodies and the advanced ECL detection system (Amersham Biosciences).
3
Immunoprecipitation of Schistosoma Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation of SmHDAC8, SmDia-RBD and SmRho1 (isoforms and mutant constructs) proteins expressed in oocytes was performed using respectively HA and Myc tags. 15h after cRNA injection in the equatorial region, oocytes were lysed in buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 0.05% SDS, 0.5% Triton X100, 5 mM MgCl2, 1 mg. mL -1 bovine serum albumin, 10 mg. mL -1 leupeptin, 10 mg. mL -1 aprotinin, 10 mg. mL -1 soybean trypsin inhibitor, 10 mg. mL -1 benzamidine, 1 mM sodium vanadate) and centrifuged at 4°C for 15 min at 10 000 xG. Supernatants were incubated with anti-Myc (1/100; Invitrogen) and anti-HA (1/100 Invitrogen) antibodies for 4 h at 4°C. Protein A-Sepharose beads (5 mg, Amersham Biosciences) were added for 1 h at 4°C. Immune complexes were collected by centrifugation, rinsed three times, resuspended in Laemmli sample buffer, and subjected to a 10% SDS-PAGE. Immune complexes were analyzed by Western blotting using anti-Myc (1/50 000) or anti-HA (1/10 000) antibodies and the advanced ECL detection system (Amersham Biosciences).
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