ubc13 ube2n  (Proteintech)

Journal: STAR protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: Figure 5. Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 mL), cell pellet (P) (6 mL), cell lysate supernatant (S) (3 mL), Ni-NTA resin flowthrough (FT) (3 mL), and Ni-NTA elution fractions (lanes 6–11) (3 mL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 mL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 mL) were analyzed by SDS-PAGE and Coomassie staining. (D) SDS-PAGE of purified E2s (UBE2D3, UBE2W, UBE2N, UBE2E3, UBE2V2, UBE2E2, UBE2E1, UBE2B), Lane 1 shows MW markers.

Article Snippet: The plasmids of pFastbac-Flag-BRCA1 (modified based on the plasmid from Jeffrey Parvin; deposited in Addgene (#223228)) and pFastBac-TwinStrepTag-BARD1 (Addgene#137166) are used for Flag-BRCA1 and Twin-StrepTag-BARD1 expression in insect cells.8,9 c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a- UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24aUBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before.5,10–12 Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.13 Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/ UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.13

Techniques: SDS Page, Staining, Chromatography, Size-exclusion Chromatography

Journal: STAR protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: Figure 6. In vitro ubiquitylation of histone H2A by BRCA1-BARD1 and its variants (A) Schematic of ubiquitination assay mediated by E3 BRCA1-BARD1 with NCP as a substrate. (B) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency of BRCA1-BARD1 in conjunction with different E2s. UBE2B that does not bind BRCA1-BARD1 was included as a negative control. BC1-BD1, BRCA1-BARD1. (C) The BRCA1-BARD1 RING structure showing the locations of BRCA1 residues I26, L63, and K65 with the E2 binding surface indicated by dotted lines (PDB:7JZV). (D) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency for wild-type or mutant BRCA1-BARD1 in conjunction with NCP as a substrate and E2s: UBE2D3 (top) and UBE2D3 + UBE2N-UBE2V2 (bottom).

Article Snippet: The plasmids of pFastbac-Flag-BRCA1 (modified based on the plasmid from Jeffrey Parvin; deposited in Addgene (#223228)) and pFastBac-TwinStrepTag-BARD1 (Addgene#137166) are used for Flag-BRCA1 and Twin-StrepTag-BARD1 expression in insect cells.8,9 c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a- UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24aUBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before.5,10–12 Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.13 Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/ UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.13

Techniques: In Vitro, Ubiquitin Proteomics, Negative Control, Binding Assay, Mutagenesis