In vitro de-2-hydroxyisobutyrylation, deacetylation, and desuccinylation activity assay for CobB using whole-protein lysate
This protocol is extracted from research article:
Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes
Sci Adv, Jul 17, 2019; DOI: 10.1126/sciadv.aaw6703

The assay was performed as described in our previous study (3). Briefly, P. mirabilis strain ATCC29906 was cultured overnight in LB medium at 37°C and then harvested and extracted during the exponential growth phase by centrifugation. Protein lysates (10 μg) were resolved by SDS-PAGE gel and transferred to the polyvinylidene difluoride membranes. The membranes were incubated with or without 50 μg of CobB in 2 ml of reaction buffer [50 mM tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM NAD+, and 0.1% PEG-8000 (polyethylene glycol, molecular weight 8000) (pH 8.0)] at 37°C overnight. Then, the membranes were blocked and probed with anti-Khib antibody, anti-Kac antibody, and anti-Ksucc antibody, respectively. Coomassie blue staining was used as a loading control.

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