Anti-TIMP3 antibodies for immunofluorescent immunohistochemistry were purchased from Novus Biologicals (Littleton, CO, USA). Anti-FLAG and anti-myc antibodies were from Sigma-Aldrich Corp. (St. Louis, MO, USA); anti-HA-tag antibody was from Cell Signaling Technology (Danvers, MA, USA). Polyclonal rabbit antiserum against mouse myocilin was described previously.11 (link) Purified anti-myocilin antibodies were purified from anti-myocilin rabbit serum through an antigen-specific affinity purification. Normal rabbit IgG was purchased from Sigma-Aldrich Corp. Affinity-purified anti-myocilin antibodies were labeled with horseradish peroxidase (HRP) using the HRP conjugation kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Human myocilin, myocilin-ΔC, and myocilin-ΔN constructs tagged with the FLAG epitope were previously described.34 (link) Mouse Timp3 cDNA was cloned into the pcDNA3.1/Myc-His vector (Invitrogen, Carlsbad, CA, USA). Human full-length TIMP2, TIMP3, and TIMP4 cDNAs were cloned into the pcDNA3 vector in-frame with hemagglutinin tag (HA).
Anti flag and anti myc antibodies
Manufactured by Merck Group
Sourced in United States
Anti-Flag and anti-Myc antibodies are laboratory reagents used for the detection and purification of recombinant proteins in biochemical and molecular biology applications. These antibodies specifically recognize and bind to the Flag and Myc epitope tags, which are commonly used as fusion tags to facilitate the identification and purification of target proteins. The core function of these antibodies is to provide a reliable and sensitive tool for the detection and enrichment of tagged recombinant proteins in various experimental setups.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Glutathione sepharose 4b gel, by GE Healthcare (2 mentions)
Hrp conjugation kit, by Abcam (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
Pcdna3.1 myc his vector, by Thermo Fisher Scientific (1 mentions)
Anti ha tag antibody, by Cell Signaling Technology (1 mentions)
Pierce classic magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Anti flag m2 a nity gel, by Merck Group (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Np 40 buffer, by Beyotime (1 mentions)
Mouse anti ha monoclonal antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Semi dry transfer cell, by Bio-Rad (1 mentions)
Ecl prime western blotting substrate, by GE Healthcare (1 mentions)
Anti mouse and rabbit igg hrp conjugated secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
7 protocols using anti flag and anti myc antibodies
1
Antibody Production and Characterization for Myocilin
2
Co-immunoprecipitation Assay Protocol
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Co-IP assays were performed using Pierce Classic Magnetic IP/Co-IP Kit (Thermo Fisher) and Flag Tag IP/Co-IP Kit (Biolinkedin, China) according to the manufacturer’s instructions. Co-immunoprecipitated proteins were analyzed by western blotting as described above. Anti-Flag and anti-Myc antibodies were purchased from Sigma-Aldrich.
3
Poly(I:C) and Poly(dA:dT) Stimulation of HEK293T Cells
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HEK293T cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) in an incubator at 5% CO2 and 37°C. HMW poly(I:C) and poly(dA:dT) (In vivoGen, San Diego, CA, USA) were separately mixed with Lipofectamine 3000 (Life Technologies) at a 1:1 ratio (µg/µl) in Opti-MEM (Life Technologies) for cell stimulation. Anti-Flag and anti-Myc antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Characterization of OsWRKY62 Interactions
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The RK and KKK in the WD of OsWRKY62 (W62WD) were substituted by A residues to generate W62WDAA (mutation RK), W62WDAAA (mutation KKK), and W62WD5A (mutation of both sites), by site-directed mutagenesis. The corresponding cDNAs of OsIMαΔIBB1a, OsIMαΔIBB1b, OsWRKY62, OsWRKY76, and their mutants were inserted respectively into a modified pGEX vector (pGEX-tag: 3 × myc or 3 × flag available at the C-terminus). The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and purified 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 affinity 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 five 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 affinity 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 five 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).
5
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).
6
Influenza Polymerase Complex Interactions
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293T cells were cotransfected with Myc-tagged PB2 627E or 627K, PB1 or PB1 (D446Y), PA and NP as well as ANP32A and cNA-Luc template for 24 h. The cells were resuspended in NP-40 buffer (Beyotime) and incubated for 1 h at 4°C. Immunoprecipitations were performed with protein A/G-agarose (Santa Cruz) and 5 μg of mouse anti-NP monoclonal antibody. Immunoprecipitated proteins were washed and dissolved in SDS sample buffer, and the interaction between NP and polymerase was analyzed by western blot using mouse anti-NP monoclonal antibody and anti-Myc and anti-Flag antibodies (Sigma) for detection of PB2 and ANP32A, respectively.
7
TCA-based Whole Cell Extraction and Immunoprecipitation
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Whole cell extracts were prepared using the trichloroacetic acid (TCA) method as previously described (39 (link)). Immunoprecipitation was carried out following our published method (39 (link)). Proteins from whole-cell extracts or immunoprecipitated samples were resolved on a SDS-PAGE gel and transferred onto a PVDF membrane (Millipore) using a semi-dry transfer cell (Bio-Rad). Mouse monoclonal anti-HA antibody was purchased from Abcam, anti-Myc and anti-FLAG antibodies were purchased from Sigma, and the phosphor-specific rabbit polycolonal antibody against Fun30 phosphorylation on serine 28 was ordered from GenScript (Nanjing). Anti-mouse and rabbit IgG HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. Blots were developed using the ECL Prime Western Blotting substrate (GE Healthcare).
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