Human embryonic kidney cell lines (HEK293T from ATCC) were maintained in advanced DMEM, supplemented with Glutamine and 10% (v/v) fetal bovine serum. A day before transfection, approximately 3 × 106 cells were seeded on a 100 mm dish. Transfection was performed with a mixture of pcDNA plasmids expressing specific proteins, as indicated, using Lipofectamine 3000 (Thermo Fisher Scientific) with empty pcDNA vector plasmid as a balance. After 48 h, cells were harvested and lysed with sonication in the lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.5) with complete protease inhibitor mixture (Sigma). Some cell lysate was mixed with loading buffer and heated at 95 °C for 5 min. For immunoprecipitation, the cleared cell lysate was incubated with 20 µL of anti-FLAG magnetic beads (Sigma) with shaking at 4 °C for 5 h. The beads were washed with the lysis buffer three times and bound proteins were eluted with FLAG peptides at 0.1 mg/mL. Samples were subjected to SDS-PAGE and Western Blotting analysis with appropriate antibodies. Antibodies used in the currernt study included anti-HA (COVANCE), anti-FLAG (Abnova), anti-DDB1 (Sigma), anti-Actin (Sigma), anti-DCAF1 (Santa Cruz), anti-SIRT7 (Santa Cruz), anti-Goat IgG (Santa Cruz), anti-Rabbit IgG (Sigma), and anti-Mouse IgG (Sigma).
Anti dcaf1
Manufactured by Santa Cruz Biotechnology
Anti-DCAF1 is a laboratory reagent used for research purposes. It is designed to detect and analyze the DCAF1 protein, which plays a role in various cellular processes. The product provides a tool for researchers to study the expression, localization, and interactions of DCAF1 in their experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Complete protease inhibitor mixture, by Merck Group (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti sirt7, by Santa Cruz Biotechnology (2 mentions)
Anti ddb1, by Merck Group (2 mentions)
Anti flag, by Abnova (2 mentions)
Anti flag magnetic beads, by Merck Group (2 mentions)
Anti ha, by Fortrea (2 mentions)
96 well glass bottom plate, by Cellvis (1 mentions)
Hoescht 33342, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti dcaf1
1
Co-immunoprecipitation of proteins in HEK293T cells
2
Visualizing TMPRSS2-DCAF1 Interactions
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Proximity Ligation Assay was conducted using Duolink technology, according to manufacturer’s protocol (Millipore-Sigma)62 (link). Briefly, Beas-2B cells were seeded to 96-well glass bottom plate (Cellvis) and fixed with 4% paraformaldehyde for 1 h. Cells were permeabilized with 0.5% Triton-X-100 for 0.5 h, and blocked with Duolink Blocking solution at 37 °C for 2 h. Anti-TMPRSS2 (Thermo, PA5-14264) and anti-DCAF1 (Santa Cruz Biotechnology, (C-8, sc-376850) antibodies were incubated with cells overnight. PLA probe incubation (secondary antibody), Ligation, and Amplification were conducted according to protocol. Cells were counterstained with Hoescht 33342 (Invitrogen). Representative confocal images were taken using Leica SP8 confocal.
3
Immunoprecipitation and Western Blot Analysis of Protein Complexes in HEK293T Cells
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Human embryonic kidney cell lines (HEK293T from ATCC) were maintained in advanced DMEM, supplemented with Glutamine and 10% (v/v) fetal bovine serum. A day before transfection, approximately 3 x 10 6 cells were seeded on a 100 mm dish. Transfection was performed with a mixture of pcDNA plasmids expressing specific proteins, as indicated, using Lipofectamine 3000 (Thermo Fisher Scientific) with empty pcDNA vector plasmid as a balance. After 48 h, cells were harvested and lysed with sonication in the lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.5) with complete protease inhibitor mixture (Sigma). Some cell lysate was mixed with loading buffer and heated at 95 o C for 5 min. For immunoprecipitation, the cleared cell lysate was incubated with 20 µL of anti-FLAG magnetic beads (Sigma) with shaking at 4 °C for 5 hours. The beads were washed with the lysis buffer three times and bound proteins were eluted with FLAG peptides at 0.1 mg/mL. Samples were subjected to SDS-PAGE and Western Blotting analysis with appropriate antibodies. Antibodies used in the currernt study included anti-HA (COVANCE), anti-FLAG (Abnova), anti-DDB1 (Sigma), anti-Actin (Sigma), anti-DCAF1 (Santa Cruz), anti-SIRT7 (Santa Cruz), anti-Goat IgG (Santa Cruz), anti-Rabbit IgG (Sigma), and anti-Mouse IgG (Sigma).
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