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ubch5c wt pet28a  (Addgene inc)


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    Addgene inc ubch5c wt pet28a
    a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, <t>UBCH5C,</t> Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.
    Ubch5c Wt Pet28a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ubch5c wt pet28a/product/Addgene inc
    Average 93 stars, based on 19 article reviews
    ubch5c wt pet28a - by Bioz Stars, 2026-03
    93/100 stars

    Images

    1) Product Images from "Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates"

    Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

    Journal: Nature Structural & Molecular Biology

    doi: 10.1038/s41594-024-01457-6

    a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.
    Figure Legend Snippet: a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, SDS Page, Staining, Activity Assay, Western Blot, Ubiquitin Proteomics, Concentration Assay



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    ubch5c wt pet28a  (Addgene inc)
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    Addgene inc ubch5c wt pet28a
    a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, <t>UBCH5C,</t> Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.
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    pet3a huba1  (Addgene inc)
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    a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, <t>UBCH5C,</t> Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.
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    e2s  (Addgene inc)
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    Purification of <t>UBE2D3</t> and other <t>E2s</t> (A) Whole cell lysate (WCL) (6 μL), cell pellet (P) (6 μL), cell lysate supernatant (S) (3 μL), Ni-NTA resin flowthrough (FT) (3 μL), and Ni-NTA elution fractions (lanes 6–11) (3 μL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 μL) 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.
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    Image Search Results


    a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

    Journal: Nature Structural & Molecular Biology

    Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

    doi: 10.1038/s41594-024-01457-6

    Figure Lengend Snippet: a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

    Article Snippet: UbcH5c WT pET28a was a gift from Rachel Klevit (Addgene plasmid #12643; http://n2t.net/addgene:12643 ; RRID: Addgene_12643 ) . pET3a-hUBA1 was a gift from Titia Sixma (Addgene plasmid # 63571; http://n2t.net/addgene:63571 ; RRID: Addgene_63571 ) .

    Techniques: Sequencing, Agarose Gel Electrophoresis, SDS Page, Staining, Activity Assay, Western Blot, Ubiquitin Proteomics, Concentration Assay

    Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 μL), cell pellet (P) (6 μL), cell lysate supernatant (S) (3 μL), Ni-NTA resin flowthrough (FT) (3 μL), and Ni-NTA elution fractions (lanes 6–11) (3 μL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 μL) 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.

    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: Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 μL), cell pellet (P) (6 μL), cell lysate supernatant (S) (3 μL), Ni-NTA resin flowthrough (FT) (3 μL), and Ni-NTA elution fractions (lanes 6–11) (3 μL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 μL) 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., c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a-UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24a-UBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before., , , Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.

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

    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).

    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: 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., c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a-UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24a-UBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before., , , Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.

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