Quantifying Protein Sulfenylation via DCP-Bio1

To measure sulfenic acid (CysOH) formation (sulfenylation) of proteins, cells were lysed in degassed-specific lysis buffer [50 mM HEPES, pH7.0 at room temperature, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 10 mM sodium pyrophosphate, 5 mM Iodoacetamide (IAA), 100 μM DTPA, 1% Triton-X-100, protease inhibitor, 200 unit/mL catalase (Calbiochem), 200 μM DCP-Bio1 (KaraFast, USA)] and then DCP-Bio1-bound proteins were pulled down with streptavidin beads (Thermo scientific, USA) overnight at 4 °C. DCP-Bio1-bound CysOH formed, sulfenylated-proteins were determined by immunoblotting with specific antibodies, as reported ^{19 (link), 40 (link), 61 (link)}.

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Das A., Ash D., Fouda A.Y., Sudhahar V., Kim Y.M., Hou Y., Hudson F.Z., Stansfield B.K., Caldwell R.B., McMenamin M., Littlejohn R., Su H., Regan M.R., Merrill B.J., Poole L.B., Kaplan J.H., Fukai T, & Ushio-Fukai M. (2022). Cysteine Oxidation of Copper transporter SLC31A1/CTR1, drives VEGFR2 signaling and Angiogenesis. Nature cell biology, 24(1), 35-50.

Publication 2021

DCP-Bio1 streptavidin beads

Antibodies Buffer Catalase Cells Dtpa Edta Hepes Iodoacetamide Nacl Protease inhibitor Proteins Sodium pyrophosphate Streptavidin Sulfenic acid Triton x 100

Corresponding Organization :

Other organizations : Augusta University, University of Illinois at Chicago, Illinois College, University of Illinois Urbana-Champaign, Wake Forest University

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Variable analysis

independent variables

Lysis buffer composition: 50 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 5 mM Iodoacetamide (IAA), 100 μM DTPA, 1% Triton-X-100, protease inhibitor, 200 unit/mL catalase (Calbiochem), 200 μM DCP-Bio1 (KaraFast, USA)

dependent variables

Formation of sulfenylated proteins (CysOH) determined by immunoblotting with specific antibodies

control variables

Cell lysis performed in degassed-specific lysis buffer
Incubation of DCP-Bio1-bound proteins with streptavidin beads overnight at 4 °C

positive controls

Not specified

negative controls

Not specified

Annotations

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