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Addgene inc pegfp blm
Pegfp Blm, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 2 article reviews

pegfp blm - by Bioz Stars, 2026-03

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BLM amino acids 1332–1349 are required for binding to topoisomerase I. ( A ) Diagram of the full-length BLM protein (1417 amino acids) highlighting known functional domains and the interaction regions with topoisomerase I (TOP1), topoisomerase IIα (TOP2A) and topoisomerase IIIα (TOP3A). An additional TOP3A-interaction region consisting of amino acids 1266–1417 was reported in one study ; ( B ) 35 S-methionine-labeled BLM peptide fragments of the indicated amino acids were generated using IVTT, incubated with purified TOP1 and immunoprecipitated using α-TOP1 or isotype control antibodies with Dynabeads Protein G. The C-terminus of BLM (amino acids 997–1417) was used as a positive control and luciferase as a negative control for each replicate. Pulldown of each peptide fragment was quantified and non-specific pulldown subtracted. Each fragment was normalized to its input and expressed as % relative IP compared to the positive control (amino acids 997–1417). Results from 3–9 experiments per peptide were compared to the positive control 997–1417 fragment using a Student’s t -test. A representative gel is shown for each. Error bars depict standard deviation. * p < 0.006, ** p < 0.001.

Journal: Genes

Article Title: Regulation of BLM Nucleolar Localization

doi: 10.3390/genes7090069

Figure Lengend Snippet: BLM amino acids 1332–1349 are required for binding to topoisomerase I. ( A ) Diagram of the full-length BLM protein (1417 amino acids) highlighting known functional domains and the interaction regions with topoisomerase I (TOP1), topoisomerase IIα (TOP2A) and topoisomerase IIIα (TOP3A). An additional TOP3A-interaction region consisting of amino acids 1266–1417 was reported in one study ; ( B ) 35 S-methionine-labeled BLM peptide fragments of the indicated amino acids were generated using IVTT, incubated with purified TOP1 and immunoprecipitated using α-TOP1 or isotype control antibodies with Dynabeads Protein G. The C-terminus of BLM (amino acids 997–1417) was used as a positive control and luciferase as a negative control for each replicate. Pulldown of each peptide fragment was quantified and non-specific pulldown subtracted. Each fragment was normalized to its input and expressed as % relative IP compared to the positive control (amino acids 997–1417). Results from 3–9 experiments per peptide were compared to the positive control 997–1417 fragment using a Student’s t -test. A representative gel is shown for each. Error bars depict standard deviation. * p < 0.006, ** p < 0.001.

Article Snippet: GM08505 cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids (pEGFP-BLM-wild-type, pEGFP-BLM-D795A, pEGFP-BLM-S1342A/S1345A, pEGFP-BLM-S1342D/S1345D, pEGFP-BLM-S1342A, pEGFP-BLM-S1345A, pEGFP-BLM-S1342D, pEGFP-BLM-S1345D and pEGFP-BLM-Δ1332–1349) with Lipofectamine2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Binding Assay, Functional Assay, Labeling, Generated, Incubation, Purification, Immunoprecipitation, Positive Control, Luciferase, Negative Control, Standard Deviation

Unwinding of RNA:DNA substrate by BLM-Δ1332–1349 is not stimulated by TOP1. Kinetics of unwinding of RNA:DNA substrate was assessed using a range of BLM protein concentrations with and without TOP1. Representative gels and kinetics of unwinding for one representative protein concentration, 0.2 nM BLM-WT ( A ) or 0.3 nM BLM-Δ1332–1349 ( B ) are shown. BLM protein with two-fold molar excess TOP1 or equivalent volume of reaction buffer was incubated with 2 fmol RNA:DNA substrate for 0.25, 0.5, 0.75, 1, 2, 3, 7 and 12 min at 37 °C, and double- and single-stranded products were separated on non-denaturing acrylamide gels. Heat denatured (HD) substrate was generated by heating at 95 °C for 5 min. Unwinding was quantified as the amount of single-stranded substrate generated compared to the total substrate per time-point. Experiments were repeated 3 to 6 times for each protein concentration, and curves showing amount of substrate unwound (fmol) as a function of time were fitted to hyperbolic plots corresponding to the Michaelis-Menten equation. Reactions with TOP1 alone were performed with each experiment to verify the lack of unwinding ability of TOP1. Error bars depict standard deviation. ( C ) Specific activities of BLM proteins with and without TOP1 were calculated by measuring initial unwinding rates for 4 protein concentrations (as in panels A and B; ) and graphed as a function of protein concentration; specific activity was calculated from the slope of the line (fmol substrate unwound/min/nM protein). Error bars depict standard deviation.

Journal: Genes

Article Title: Regulation of BLM Nucleolar Localization

doi: 10.3390/genes7090069

Figure Lengend Snippet: Unwinding of RNA:DNA substrate by BLM-Δ1332–1349 is not stimulated by TOP1. Kinetics of unwinding of RNA:DNA substrate was assessed using a range of BLM protein concentrations with and without TOP1. Representative gels and kinetics of unwinding for one representative protein concentration, 0.2 nM BLM-WT ( A ) or 0.3 nM BLM-Δ1332–1349 ( B ) are shown. BLM protein with two-fold molar excess TOP1 or equivalent volume of reaction buffer was incubated with 2 fmol RNA:DNA substrate for 0.25, 0.5, 0.75, 1, 2, 3, 7 and 12 min at 37 °C, and double- and single-stranded products were separated on non-denaturing acrylamide gels. Heat denatured (HD) substrate was generated by heating at 95 °C for 5 min. Unwinding was quantified as the amount of single-stranded substrate generated compared to the total substrate per time-point. Experiments were repeated 3 to 6 times for each protein concentration, and curves showing amount of substrate unwound (fmol) as a function of time were fitted to hyperbolic plots corresponding to the Michaelis-Menten equation. Reactions with TOP1 alone were performed with each experiment to verify the lack of unwinding ability of TOP1. Error bars depict standard deviation. ( C ) Specific activities of BLM proteins with and without TOP1 were calculated by measuring initial unwinding rates for 4 protein concentrations (as in panels A and B; ) and graphed as a function of protein concentration; specific activity was calculated from the slope of the line (fmol substrate unwound/min/nM protein). Error bars depict standard deviation.

Techniques: Protein Concentration, Incubation, Generated, Standard Deviation, Activity Assay

The TOP1-interaction domain of BLM is highly conserved in mammals. BLM protein sequences were extracted from Ensembl and aligned using ClustalW 1.83 at the European Bioinformatics Institute. The output was re-formatted in MView, using default parameters. Identities are normalized by aligned length. Residues are colored by identity and property. The TOP1-interaction region is indicated with a black bar and corresponds to human BLM residues 1332–1349. Serines 1342 and 1345 are highlighted with arrows. The nuclear localization sequence (NLS) region of BLM is contained within this region (1334–1349). The number in brackets at the end of each entry indicates percentage identity of the full-length protein sequence with respect to the human BLM protein. Consensus sequences for 90% and 100% identity are shown below. Accession numbers for the sequences are as follows: human [ Homo sapiens ; NP_000048.1]; bonobo [ Pan paniscus ; XP_003807768.1]; chimpanzee [ Pan troglodytes ; XP_510594.2]; lowland gorilla [ Gorilla gorilla gorilla ; XP_004056835.1]; drill [ Mandrillus leucophaeus ; XP_011833777.1]; marmoset [ Callithrix jacchus ; XP_002749167.1]; grey mouse lemur [ Microcebus murinus ; XP_012618727.1]; killer whale [ Orcinus orca ; XP_004278332.1]; horse [ Equus caballus ; XP_001502766.1]; Bactrian camel [ Camelus ferus ; XP_006192661.1]; bison [ Bison bison bison ; XP_010839366.1]; cow [ Bos Taurus ; XP_613809.3]; domestic dog [ Canis lupus familiaris ; XP_003434427.1]; domestic cat [ Felis catus ; XP_011281067.1]; polar bear [ Ursus maritimus ; XP_008686016.1]; sheep [ Ovis aries ; XP_004017810.1]; chinchilla [ Chinchilla lanigera ; XP_005381562.1]; mouse [ Mus musculus ; NP_031576.4]; rat [ Rattus norvegicus ; XP_003753349.2].

Journal: Genes

Article Title: Regulation of BLM Nucleolar Localization

doi: 10.3390/genes7090069

Figure Lengend Snippet: The TOP1-interaction domain of BLM is highly conserved in mammals. BLM protein sequences were extracted from Ensembl and aligned using ClustalW 1.83 at the European Bioinformatics Institute. The output was re-formatted in MView, using default parameters. Identities are normalized by aligned length. Residues are colored by identity and property. The TOP1-interaction region is indicated with a black bar and corresponds to human BLM residues 1332–1349. Serines 1342 and 1345 are highlighted with arrows. The nuclear localization sequence (NLS) region of BLM is contained within this region (1334–1349). The number in brackets at the end of each entry indicates percentage identity of the full-length protein sequence with respect to the human BLM protein. Consensus sequences for 90% and 100% identity are shown below. Accession numbers for the sequences are as follows: human [ Homo sapiens ; NP_000048.1]; bonobo [ Pan paniscus ; XP_003807768.1]; chimpanzee [ Pan troglodytes ; XP_510594.2]; lowland gorilla [ Gorilla gorilla gorilla ; XP_004056835.1]; drill [ Mandrillus leucophaeus ; XP_011833777.1]; marmoset [ Callithrix jacchus ; XP_002749167.1]; grey mouse lemur [ Microcebus murinus ; XP_012618727.1]; killer whale [ Orcinus orca ; XP_004278332.1]; horse [ Equus caballus ; XP_001502766.1]; Bactrian camel [ Camelus ferus ; XP_006192661.1]; bison [ Bison bison bison ; XP_010839366.1]; cow [ Bos Taurus ; XP_613809.3]; domestic dog [ Canis lupus familiaris ; XP_003434427.1]; domestic cat [ Felis catus ; XP_011281067.1]; polar bear [ Ursus maritimus ; XP_008686016.1]; sheep [ Ovis aries ; XP_004017810.1]; chinchilla [ Chinchilla lanigera ; XP_005381562.1]; mouse [ Mus musculus ; NP_031576.4]; rat [ Rattus norvegicus ; XP_003753349.2].

Techniques: Sequencing

S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p < 0.0001).

Journal: Genes

Article Title: Regulation of BLM Nucleolar Localization

doi: 10.3390/genes7090069

Figure Lengend Snippet: S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p < 0.0001).

Techniques: Transfection, Staining, Standard Deviation

Unwinding of RNA:DNA substrate by BLM-S1342A/S1345A or BLM-S1342D/S1345D is equally stimulated by TOP1. Kinetics of unwinding of RNA:DNA substrate were assessed using a range of BLM protein concentrations with and without TOP1. Representative gels and kinetics of unwinding for one representative protein concentration, 0.2 nM, by BLM-S1342A/S1345A ( A ) or BLM-S1342D/S1345D ( B ) are shown. BLM protein with two-fold molar excess of TOP1 or equivalent volume of reaction buffer was incubated with 2 fmol RNA:DNA substrate for 0.25, 0.5, 0.75, 1, 2, 3, 7 and 12 min at 37 °C; double- and single-stranded products were separated on non-denaturing acrylamide gels. Heat denatured (HD) substrate was generated by heating at 95 °C for 5 min. Unwinding was quantified as the amount of single-stranded substrate compared to the total substrate per time-point. Experiments were repeated 3 to 6 times for each protein concentration, and curves showing amount of substrate unwound (fmol) as a function of time were fitted to hyperbolic plots corresponding to the Michaelis-Menten equation. BLM-WT was used as a positive control for the ability of TOP1 to stimulate BLM . Reactions with TOP1 alone were performed with each experiment to verify the lack of unwinding ability of TOP1. Error bars depict standard deviation. ( C ) Specific activities of BLM proteins with and without TOP1 were calculated by measuring initial unwinding rates for 4 protein concentrations (as in panels A and B; ) and graphed as a function of protein concentration; specific activity was calculated from the slope of the line (fmol substrate unwound/min/nM protein) . Error bars depict standard deviation. ( D ) Table showing the fold-change of RNA:DNA unwinding activity of each BLM protein with TOP1. Specific activities shown (fmol/min/nM) were obtained as in panel C and C.

Journal: Genes

Article Title: Regulation of BLM Nucleolar Localization

doi: 10.3390/genes7090069

Figure Lengend Snippet: Unwinding of RNA:DNA substrate by BLM-S1342A/S1345A or BLM-S1342D/S1345D is equally stimulated by TOP1. Kinetics of unwinding of RNA:DNA substrate were assessed using a range of BLM protein concentrations with and without TOP1. Representative gels and kinetics of unwinding for one representative protein concentration, 0.2 nM, by BLM-S1342A/S1345A ( A ) or BLM-S1342D/S1345D ( B ) are shown. BLM protein with two-fold molar excess of TOP1 or equivalent volume of reaction buffer was incubated with 2 fmol RNA:DNA substrate for 0.25, 0.5, 0.75, 1, 2, 3, 7 and 12 min at 37 °C; double- and single-stranded products were separated on non-denaturing acrylamide gels. Heat denatured (HD) substrate was generated by heating at 95 °C for 5 min. Unwinding was quantified as the amount of single-stranded substrate compared to the total substrate per time-point. Experiments were repeated 3 to 6 times for each protein concentration, and curves showing amount of substrate unwound (fmol) as a function of time were fitted to hyperbolic plots corresponding to the Michaelis-Menten equation. BLM-WT was used as a positive control for the ability of TOP1 to stimulate BLM . Reactions with TOP1 alone were performed with each experiment to verify the lack of unwinding ability of TOP1. Error bars depict standard deviation. ( C ) Specific activities of BLM proteins with and without TOP1 were calculated by measuring initial unwinding rates for 4 protein concentrations (as in panels A and B; ) and graphed as a function of protein concentration; specific activity was calculated from the slope of the line (fmol substrate unwound/min/nM protein) . Error bars depict standard deviation. ( D ) Table showing the fold-change of RNA:DNA unwinding activity of each BLM protein with TOP1. Specific activities shown (fmol/min/nM) were obtained as in panel C and C.

Techniques: Protein Concentration, Incubation, Generated, Positive Control, Standard Deviation, Activity Assay

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