dna fragments encoding 3x superfolder gfp Search Results


dna fragments encoding 3x superfolder gfp  (Addgene inc)
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Addgene inc dna fragments encoding 3x superfolder gfp
( A ) Representative live cell imaging of a DT40 CDK1 as _Halo-lamin B1_3xGFP-NES cell released from G 2 block with 1NM-PP1. <t>DNA:grey;</t> Halo-lamin B1: magenta; 3xGFP-NES: green. Seven z-sections (1 μm interval) were taken every 1.5 min. A single section is shown for each timepoint. Scale bar = 5 μm. 3XGFP enters nuclei a few minutes prior to visible nuclear lamina disruption (NEB, t= 10–11 min). Intensity of lines under the images illustrates the relative amount of the indicated complexes on chromatin at each time point. Green arrow indicates cytoplasmic <t>GFP</t> entering the nucleus before nuclear envelope breakdown. ( B ) Relative nuclear GFP fluorescence intensity (cytosolic GFP intensity = 1) from experiment of . ( C ) Chromatin enrichment for proteomics (ChEP) analysis of WT CDK1 as cells (SILAC analysis). Log 2 SILAC ratio normalized against G 2 is shown for cohesin (average of SMC1, SMC3, and RAD21), condensin I (CAP-H, CAP-G, and CAP-D2) and condensin II complexes (CAP-H2, CAP-G2, and CAP-D3). t= 0 is after completion of 1NM-PP1 washout, n= 6. ( D ) Estimated number of chromatin-associated cohesin, condensin I and condensin II complexes (per Mb DNA) during mitotic entry in wild type CDK1 as cells. Average iBAQ number from ChEP analysis for subunits as listed in C was normalized relative to values for Histone H4, n= 6. ( E ) Absolute quantification of SMC3 (cohesin subunit), CAP-H (condensin I subunit) and CAP-H2 (condensin II subunit) on chromatin (per Mb DNA). Protein numbers are calculated following ChEP analysis of corresponding Halo-tagged cell lines normalized using purified spike-in Halo-Histone H4 protein (n= 4).
Dna Fragments Encoding 3x Superfolder Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) Representative live cell imaging of a DT40 CDK1 as _Halo-lamin B1_3xGFP-NES cell released from G 2 block with 1NM-PP1. DNA:grey; Halo-lamin B1: magenta; 3xGFP-NES: green. Seven z-sections (1 μm interval) were taken every 1.5 min. A single section is shown for each timepoint. Scale bar = 5 μm. 3XGFP enters nuclei a few minutes prior to visible nuclear lamina disruption (NEB, t= 10–11 min). Intensity of lines under the images illustrates the relative amount of the indicated complexes on chromatin at each time point. Green arrow indicates cytoplasmic GFP entering the nucleus before nuclear envelope breakdown. ( B ) Relative nuclear GFP fluorescence intensity (cytosolic GFP intensity = 1) from experiment of . ( C ) Chromatin enrichment for proteomics (ChEP) analysis of WT CDK1 as cells (SILAC analysis). Log 2 SILAC ratio normalized against G 2 is shown for cohesin (average of SMC1, SMC3, and RAD21), condensin I (CAP-H, CAP-G, and CAP-D2) and condensin II complexes (CAP-H2, CAP-G2, and CAP-D3). t= 0 is after completion of 1NM-PP1 washout, n= 6. ( D ) Estimated number of chromatin-associated cohesin, condensin I and condensin II complexes (per Mb DNA) during mitotic entry in wild type CDK1 as cells. Average iBAQ number from ChEP analysis for subunits as listed in C was normalized relative to values for Histone H4, n= 6. ( E ) Absolute quantification of SMC3 (cohesin subunit), CAP-H (condensin I subunit) and CAP-H2 (condensin II subunit) on chromatin (per Mb DNA). Protein numbers are calculated following ChEP analysis of corresponding Halo-tagged cell lines normalized using purified spike-in Halo-Histone H4 protein (n= 4).

Journal: Science (New York, N.Y.)

Article Title: Rules of engagement for condensins and cohesins guide mitotic chromosome formation

doi: 10.1126/science.adq1709

Figure Lengend Snippet: ( A ) Representative live cell imaging of a DT40 CDK1 as _Halo-lamin B1_3xGFP-NES cell released from G 2 block with 1NM-PP1. DNA:grey; Halo-lamin B1: magenta; 3xGFP-NES: green. Seven z-sections (1 μm interval) were taken every 1.5 min. A single section is shown for each timepoint. Scale bar = 5 μm. 3XGFP enters nuclei a few minutes prior to visible nuclear lamina disruption (NEB, t= 10–11 min). Intensity of lines under the images illustrates the relative amount of the indicated complexes on chromatin at each time point. Green arrow indicates cytoplasmic GFP entering the nucleus before nuclear envelope breakdown. ( B ) Relative nuclear GFP fluorescence intensity (cytosolic GFP intensity = 1) from experiment of . ( C ) Chromatin enrichment for proteomics (ChEP) analysis of WT CDK1 as cells (SILAC analysis). Log 2 SILAC ratio normalized against G 2 is shown for cohesin (average of SMC1, SMC3, and RAD21), condensin I (CAP-H, CAP-G, and CAP-D2) and condensin II complexes (CAP-H2, CAP-G2, and CAP-D3). t= 0 is after completion of 1NM-PP1 washout, n= 6. ( D ) Estimated number of chromatin-associated cohesin, condensin I and condensin II complexes (per Mb DNA) during mitotic entry in wild type CDK1 as cells. Average iBAQ number from ChEP analysis for subunits as listed in C was normalized relative to values for Histone H4, n= 6. ( E ) Absolute quantification of SMC3 (cohesin subunit), CAP-H (condensin I subunit) and CAP-H2 (condensin II subunit) on chromatin (per Mb DNA). Protein numbers are calculated following ChEP analysis of corresponding Halo-tagged cell lines normalized using purified spike-in Halo-Histone H4 protein (n= 4).

Article Snippet: DNA fragments encoding 3x superfolder GFP or mCherry (Addgene plasmids 75385 and 75387 digested with BamHI/XhoI) and double-stranded oligos encoding BP-NLS or NES from Gg cAMP-dependent kinase inhibitor alpha were ligated into pcDNA3 (digested with BamHI/ApaI).

Techniques: Live Cell Imaging, Blocking Assay, Disruption, Fluorescence, Multiplex sample analysis, Quantitative Proteomics, Purification