Polyhistidine-tagged UBE2D1 (UbcH5a) and Glutathione-S-transferase (GST)-tagged ICP0.241 (amino acids 1–241 of ICP0 encompassing the C3HC4 RING-finger domain) were purified from bacterial extracts using affinity isolation chromatography, as described previously [6] (link). Polyhistidine-tagged E1 ubiquitin-activating enzyme was purified from baculovirus-infected cell extracts, as described previously [6] (link). Wild-type ubiquitin (Sigma–Aldrich; U6253) and methylated-ubiquitin (BostonBiochem; U-501) were purchased from commercial sources.
Wild type ubiquitin
Manufactured by Merck Group
Wild-type ubiquitin is a small, highly conserved protein that plays a crucial role in cellular processes. It functions as a post-translational modification that targets proteins for degradation or other cellular functions. The core function of wild-type ubiquitin is to covalently attach to target proteins, marking them for destruction by the proteasome or regulating their activities within the cell.
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Lab products found in correlation
3 protocols using wild type ubiquitin
1
Purification of Ubiquitin Enzymes
2
Unanchored K63-linked Polyubiquitin Chains
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Unanchored K63-linked polyubiquitin chains were prepared by incubating 0.05 µM E1 (HisUba1), 2 µM HisUbc13-Mms2 and 0.5 µM E3 (Pib1RING+100aa)48 (link) in a 1 ml reaction containing 40 mM HEPES, pH 7.4, 8 mM magnesium acetate, 50 mM NaCl and 30 µM ATP. Wildtype ubiquitin (purified bovine ubiquitin, Sigma) was used at a concentration of 8 µM and 4 µM of ubiquitin mutant K63R was added for capping of the chains. The reaction was incubated for 1.5 h at 30 °C and 1–4 µl of the chain reaction were used in GST-pulldown assays.
3
Structural Analysis of Proteins with CD Spectroscopy
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Either 0.5 mg/ml wild type ubiquitin (Sigma) or 0.2 mg/ml Pol beta K61Plk were mixed in 25 mM Tris HCl, pH 7.5, 50 mM NaCl with different concentrations of SDS or NLS. After incubation at 4 °C for 1 h, far UV CD spectra from 250 to 200 nm were recorded using a Jasco J 815 CD spectrometer. To additionally measure a spectrum of fully denatured ubiquitin and Pol beta, the proteins were mixed with 30 mM SDS and heated for 5 min at 95 °C before measurement.
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