Anti gapdh 6c5

Cells labeled with DYn-2, which was prepared as described by Paulsen et al.,^{22 (link)} were washed three times with ice-cold PBS, then lysed with mild detergent buffer described in Immunoblotting for 20 min, and centrifuged at 4 °C and 12000g for 10 min. The protein supernatant was normalized to 1.5 mg/mL and precleared of endogenous biotin by agitation in a 150 μL slurry of Pierce NeutrAvidin agarose (N-agarose, Life Technologies). Cleared samples were labeled with biotin using copper-catalyzed azide alkyne cycloaddition (CuAAC) by agitation for 1 h in a buffer containing the following (final concentrations): biotin azide (0.2 mM), TBTA (0.1 mM), Cu₂SO₄ (1.0 mM), and SA (1.0 mM). An aliquot of each sample was immunoblotted for total sulfenylation probed against Pierce High Sensitivity NeutrAvidin-HRP (N-HRP, Life Technologies). The remaining post-CuAAC sample was separated into equal aliquots for immunoprecipitation overnight at 4 °C with anti-GAPDH (6C5) (Santa Cruz) or anti-PTP1B (H-135) (Santa Cruz) followed by rotation for 2 h with Protein A-agarose. Samples were then prepared for immunoblotting and probed against N-HRP (Life Technologies), and anti-GAPDH (6C5) or anti-PTP1B (H-135) (both from Santa Cruz).