Reagents used in this study were as follows: Earle's balanced salt solution (Sigma, E2888), LysoTracker™ Red (Invitrogen, L7528), Ba lomycin A1 (Sigma, 19148), MG132 (MCE, HY-13259), chloroquine (Sigma, C6628), ANTI-FLAG M2 A nity Gel (Sigma, A2220), Anti-HA A nity Gel (Sigma, A2095), Glutathione Sepharose® 4B glutathione-sepharose Resin (GE, 17 Cell culture and transfection HEK293T, U2OS and Hela cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 1% Penicillin-Streptomycin Solution (Beyotime) at 37℃ with 5% CO2. For starvation treatment, cells were washed three times with PBS (Hyclone) and then incubated with EBSS for the indicated time. Hela cells were transiently transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Transient transfection of plasmid in HEK293T cells was performed using PEI according to the manufacturer's protocol . Cells were analyzed 24 h after transfection. For RNA interference, siRNA duplexes were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions, 48h after transfection, cells were harvested for analysis.
Anti flag m2 a nity gel
Manufactured by Merck Group
ANTI-FLAG M2 Affinity Gel is a lab equipment product that is designed for the purification of FLAG-tagged proteins. It consists of an agarose-based resin with immobilized anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG epitope tag. The gel can be used in chromatographic applications to isolate and purify FLAG-tagged proteins from complex samples.
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Lab products found in correlation
Ba lomycin a1, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Earle s balanced salt solution, by Merck Group (1 mentions)
Glutathione sepharose 4b gel, by GE Healthcare (1 mentions)
Anti flag and anti myc antibodies, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
2 protocols using anti flag m2 a nity gel
1
Cellular Autophagy Regulation Assay
2
Protein Interactions via Pull-down Assay
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The RK and KKK in the WD of OsWRKY62 (W62WD) were substituted by A residues to generate W62WD AA (mutation RK), W62WD AAA (mutation KKK), and W62WD 5A (mutation of both sites). The corresponding cDNAs of OsIMαΔIBB1a, OsIMαΔIBB1b, OsWRKY62, OsWRKY76, and their mutants were inserted respectively into a modi ed pGEX vector (pGEX-tag: 3×myc or 3× ag available at the C-terminus). The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and puri ed using Glutathione Sepharose 4B gel (GE Healthcare).
For pull-down assays, proper combinations of the recombinant proteins (1 µg each) were incubated with anti-Flag M2 a nity gel (Sigma) in IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% protease inhibitor cocktail (Sigma)) for 3 h at 4°C. The beads were washed ve times with the IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) and then resuspended in 2× SDS-PAGE loading buffer. The immunocomplexes were separated on 10% polyacrylamide gels and probed with anti-Flag and anti-Myc antibodies (Sigma).
For pull-down assays, proper combinations of the recombinant proteins (1 µg each) were incubated with anti-Flag M2 a nity gel (Sigma) in IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% protease inhibitor cocktail (Sigma)) for 3 h at 4°C. The beads were washed ve times with the IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) and then resuspended in 2× SDS-PAGE loading buffer. The immunocomplexes were separated on 10% polyacrylamide gels and probed with anti-Flag and anti-Myc antibodies (Sigma).
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