Citrulline-specific probe-biotin

Guidelines (The following are our recommended protocols. This protocol is only a guideline and should be modified according to your specific needs).
In vitro citrullination experiment^[1]
1. Incubate 0.5 μg of recombinant human RUNX2 protein (rhRUNX2) and 1 unit of recombinant human PAD2 with reaction buffer (50 mM HEPES (pH 7.5), 5 mM DTT, 10 mM CaCl2, 50 mM NaCl) at 37 °C for 2 hours.
2. Incubate 19.5 μL of sample (0.5 μg) with 20% trichloroacetic acid (5 μL 100% TCA) and 0.1 mM Citrulline-specific probe-biotin (0.5 μL 5 mM stock solution) at 37 °C for 30 minutes.
3. After incubation for 30 minutes, the reaction was terminated by adding citrulline dissolved in 50 mM HEPES at pH 7.6 (final concentration 100 mM).
4. The reaction mixture was placed on ice for 30 minutes, then centrifuged at 4°C to precipitate the protein.
5. The supernatant was removed, the protein precipitate was washed twice with cold acetone, and dried.
6. The protein was resuspended in neutral resuspension buffer (50 mM HEPES, pH 8.0, containing 100 mM arginine) for subsequent analysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.