Stable inducible C99-expressing HEK293 cells were transiently transfected with a myc-tagged SorLA construct using Lipofectamine 2000 and treated or not with D6, as described above. Cells were lysed in RIPA buffer and 500 μg lysates were precleared with 20 μl Protein A Sepharose (GE Healthcare) for 2 h at 4 °C. Then the supernatant was incubated for 1 h with 1 μl α-APPct antibody before adding 30 μl Protein A Sepharose beads. After several washes in RIPA buffer, the beads were resupended in SDS sample buffer (2×) and incubated for 5 min at 95 °C. 20 μl of the sample was loaded on either 8% or 12% Bio-Rad stain-free™ TGX FastCast™ acrylamide gels and subjected to western blot analysis using either an anti-myc antibody (clone 9E10, Sigma, 1:5000) or α-APPct (1:1000).
Anti myc antibody clone 9e10
Manufactured by Merck Group
The anti-myc antibody (clone 9E10) is a laboratory research tool used to detect and quantify the presence of the c-myc protein in various biological samples. It is a mouse monoclonal antibody that specifically binds to the c-myc epitope, allowing for the identification and analysis of c-myc-containing proteins.
Automatically generated - may contain errors
2 protocols using anti myc antibody clone 9e10
1
Immunoprecipitation of SorLA-APP Complex
2
Tyrosinase-Myc-Flag Protein Purification
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Cells transiently transfected with an expression construct for tyrosinase-Myc-Flag or mock transfected cells were incubated with 10 M MG132 (Sigma) or DMSO (1:1000) for 5 h at 37 • C. Cells were then harvested and lysed in lysis buffer (20 mM Tris pH 7.8, 50 mM NaCl, 10 mM MgCl 2 , 1% (v/v) NP40) including protease inhibitors (cOmplete EDTA-free, Roche) for 30 min on ice. Lysates were centrifuged at 20,000 × g and 4 • C for 15 min. Supernatants were loaded onto protein G affinity gel (Sigma) together with anti-Myc antibody (clone 9E10, Sigma) and incubated overnight at 4 • C on a rotator. The affinity gel was washed three times in high salt buffer (50 mM Tris pH 8, 650 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X-100) and three times in low salt buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X-100). Then, precipitated proteins were heated in SDS sample buffer for 5 min at 95 • C and subsequently analyzed by SDS-PAGE and western blot as described above.
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