Cells were lysed on ice in 20mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.025% SDS, 100mM NaCl, 1mM Na3VO4, 10mM NaF, and 1% protease inhibitor cocktail (Sigma). Soluble extracts were incubated for 2 h at 4C with relevant antibodies: anti-HA (Roche Applied Science), anti-V5 (Invitrogen), anti-Flag (Cell Signaling Technology) and a negative isotype control mouse immunoglobulin (IgG) (Santa Cruz Biotechnology). Immune complexes were precipitated with protein A/G agarose (Santa Cruz Biotechnology). In select experiments, cells expressing Nox5 were crosslinked with formaldehyde. Western blotting was performed as described previously [27 (link)] using anti-HA (Roche), anti-V5 (Invitrogen), anti-Hsp90 (BD Biosciences), anti-Hsp70 (Cell Signaling Technology), anti-Flag (Cell Signaling Technology), anti-p22 (Santa Cruz Biotechnology), anti-Hsp40 (Cell Signaling Technology), anti-p23 [39 (link)], anti-HOP [39 (link)], and GAPDH (Santa Cruz Biotechnology).
Negative isotype control mouse immunoglobulin igg
Manufactured by Santa Cruz Biotechnology
Negative isotype control mouse immunoglobulin (IgG) is a laboratory reagent used in flow cytometry and other immunoassays. It serves as a control to determine non-specific binding in the detection of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Anti v5, by Thermo Fisher Scientific (2 mentions)
Anti ha, by Roche (2 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti hsp70, by Cell Signaling Technology (1 mentions)
Anti p22, by Santa Cruz Biotechnology (1 mentions)
Anti hop, by Santa Cruz Biotechnology (1 mentions)
Anti hsp40, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti hsp90, by BD (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti mek, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
2 protocols using negative isotype control mouse immunoglobulin igg
1
Immunoprecipitation and Western Blotting Protocol
2
Western Blot Analysis of Protein Complexes
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Cells were lysed on ice in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 100 mM NaCl, 1 mM Na3VO4, 10 mM NaF, and 1% protease inhibitor cocktail (Sigma). Soluble extracts were incubated for 2 h at 4°C with relevant antibodies: anti-HA (Roche Applied Science) and a negative isotype control mouse immunoglobulin (IgG) (Santa Cruz Biotechnology), and complexes precipitated with protein A/G agarose (Santa Cruz Biotechnology). Western blotting was performed as described previously[22] (link), [40] (link)–[43] (link) using anti-HA (Roche), anti-V5 (Invitrogen), anti-PKCα, β, γ, ε, η, θ, ι, λ and δ (Cell Signaling Technology), anti-ERK1/2 (Cell Signaling Technology), anti-ERK1/2 phosphorylation (Cell Signaling Technology),anti-MEK(Cell Signaling Technology), and anti-GAPDH (Santa Cruz Biotechnology), and anti-Nox5 phosphorylation antibodies[21] (link).
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