Plasmid Preparation:
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation
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. .. Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17-UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.
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 .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-VSV-G Sigma-Aldrich Cat#V4888 Goat anti-rabbit IgG HRP Sigma-Aldrich Cat#A6154 Bacterial strains BL21 (DE3) competent cells Thermo Fisher Scientific Cat#C600003 BL21 (DE3) pLysS competent cells Thermo Fisher Scientific Cat#C606010 DH10EMBacY competent cells Geneva Biotech N/A Chemicals, peptides, and recombinant proteins 2-Mercaptoethanol Fisher Scientific Cat#BP176-100 Acrylamide (electrophoresis grade) Acros Organics Cat#76-06-1 Agar (granulated) Fisher Scientific Cat#BP9744-5 Ampicillin sodium salt Fisher Scientific Cat#BP1760 Aprotinin GoldBio Cat#9087-70-1 ATP GoldBio Cat#519633-61-2 Benzamidine HCl Fisher Scientific Cat#50-488-530 Bis-acrylamide Fisher Scientific Cat#BP171 Bovine serum albumin (fraction V) Fisher Scientific Cat#BP1605-100 Bromophenol blue Fisher Scientific Cat#BP115 Calcium chloride anhydrous Acros Organics Cat#10043-52-4 Chloramphenicol RPI Cat#C61000 (Continued on next page) 4 STAR Protocols 5, 103294, September 20, 2024 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chymostatin GoldBio Cat#C-315-50 Coomassie brilliant blue R250 Sigma-Aldrich Cat#1125530025 Creatine phosphate Sigma-Aldrich Cat#10621722001 Creatine phosphokinase Roche Cat#10127566001 Disodium phosphate J.T.
Expressing:
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation
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. .. Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17-UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.
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 .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-VSV-G Sigma-Aldrich Cat#V4888 Goat anti-rabbit IgG HRP Sigma-Aldrich Cat#A6154 Bacterial strains BL21 (DE3) competent cells Thermo Fisher Scientific Cat#C600003 BL21 (DE3) pLysS competent cells Thermo Fisher Scientific Cat#C606010 DH10EMBacY competent cells Geneva Biotech N/A Chemicals, peptides, and recombinant proteins 2-Mercaptoethanol Fisher Scientific Cat#BP176-100 Acrylamide (electrophoresis grade) Acros Organics Cat#76-06-1 Agar (granulated) Fisher Scientific Cat#BP9744-5 Ampicillin sodium salt Fisher Scientific Cat#BP1760 Aprotinin GoldBio Cat#9087-70-1 ATP GoldBio Cat#519633-61-2 Benzamidine HCl Fisher Scientific Cat#50-488-530 Bis-acrylamide Fisher Scientific Cat#BP171 Bovine serum albumin (fraction V) Fisher Scientific Cat#BP1605-100 Bromophenol blue Fisher Scientific Cat#BP115 Calcium chloride anhydrous Acros Organics Cat#10043-52-4 Chloramphenicol RPI Cat#C61000 (Continued on next page) 4 STAR Protocols 5, 103294, September 20, 2024 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chymostatin GoldBio Cat#C-315-50 Coomassie brilliant blue R250 Sigma-Aldrich Cat#1125530025 Creatine phosphate Sigma-Aldrich Cat#10621722001 Creatine phosphokinase Roche Cat#10127566001 Disodium phosphate J.T.
Modification:
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation
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. .. Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17-UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.
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 .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-VSV-G Sigma-Aldrich Cat#V4888 Goat anti-rabbit IgG HRP Sigma-Aldrich Cat#A6154 Bacterial strains BL21 (DE3) competent cells Thermo Fisher Scientific Cat#C600003 BL21 (DE3) pLysS competent cells Thermo Fisher Scientific Cat#C606010 DH10EMBacY competent cells Geneva Biotech N/A Chemicals, peptides, and recombinant proteins 2-Mercaptoethanol Fisher Scientific Cat#BP176-100 Acrylamide (electrophoresis grade) Acros Organics Cat#76-06-1 Agar (granulated) Fisher Scientific Cat#BP9744-5 Ampicillin sodium salt Fisher Scientific Cat#BP1760 Aprotinin GoldBio Cat#9087-70-1 ATP GoldBio Cat#519633-61-2 Benzamidine HCl Fisher Scientific Cat#50-488-530 Bis-acrylamide Fisher Scientific Cat#BP171 Bovine serum albumin (fraction V) Fisher Scientific Cat#BP1605-100 Bromophenol blue Fisher Scientific Cat#BP115 Calcium chloride anhydrous Acros Organics Cat#10043-52-4 Chloramphenicol RPI Cat#C61000 (Continued on next page) 4 STAR Protocols 5, 103294, September 20, 2024 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chymostatin GoldBio Cat#C-315-50 Coomassie brilliant blue R250 Sigma-Aldrich Cat#1125530025 Creatine phosphate Sigma-Aldrich Cat#10621722001 Creatine phosphokinase Roche Cat#10127566001 Disodium phosphate J.T.
Ubiquitin Proteomics:Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation
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. .. Note: pET15-UbE2W (Addgene#15809); pDEST17-UbE2E3 (Addgene#15789); pDEST17-UbE2E2 (Addgene#15788); pDEST17-UbE2E1 (Addgene#15787); pET21(+)-His6-Ubc13/UBE2N (Addgene#212715); MBP-His6-TEV-MMS2/UBE2V2 (Addgene#25465) are available from Addgene.
Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.
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 .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-VSV-G Sigma-Aldrich Cat#V4888 Goat anti-rabbit IgG HRP Sigma-Aldrich Cat#A6154 Bacterial strains BL21 (DE3) competent cells Thermo Fisher Scientific Cat#C600003 BL21 (DE3) pLysS competent cells Thermo Fisher Scientific Cat#C606010 DH10EMBacY competent cells Geneva Biotech N/A Chemicals, peptides, and recombinant proteins 2-Mercaptoethanol Fisher Scientific Cat#BP176-100 Acrylamide (electrophoresis grade) Acros Organics Cat#76-06-1 Agar (granulated) Fisher Scientific Cat#BP9744-5 Ampicillin sodium salt Fisher Scientific Cat#BP1760 Aprotinin GoldBio Cat#9087-70-1 ATP GoldBio Cat#519633-61-2 Benzamidine HCl Fisher Scientific Cat#50-488-530 Bis-acrylamide Fisher Scientific Cat#BP171 Bovine serum albumin (fraction V) Fisher Scientific Cat#BP1605-100 Bromophenol blue Fisher Scientific Cat#BP115 Calcium chloride anhydrous Acros Organics Cat#10043-52-4 Chloramphenicol RPI Cat#C61000 (Continued on next page) 4 STAR Protocols 5, 103294, September 20, 2024 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chymostatin GoldBio Cat#C-315-50 Coomassie brilliant blue R250 Sigma-Aldrich Cat#1125530025 Creatine phosphate Sigma-Aldrich Cat#10621722001 Creatine phosphokinase Roche Cat#10127566001 Disodium phosphate J.T.
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