nucleosomal arrays  (Worthington Biochemical)


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    Worthington Biochemical nucleosomal arrays
    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of <t>nucleosomal</t> fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .
    Nucleosomal Arrays, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    nucleosomal arrays - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "The Telomere Binding Protein TRF2 Induces Chromatin Compaction"

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019124

    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .
    Figure Legend Snippet: TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Derivative Assay

    TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.
    Figure Legend Snippet: TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.

    Techniques Used: Centrifugation, Agarose Gel Electrophoresis

    The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.
    Figure Legend Snippet: The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.

    Techniques Used: Derivative Assay, Electrophoresis, Concentration Assay

    TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.
    Figure Legend Snippet: TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.

    Techniques Used: Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, Derivative Assay, Produced

    Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .
    Figure Legend Snippet: Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .

    Techniques Used: Microscopy

    The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.
    Figure Legend Snippet: The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis

    2) Product Images from "The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure"

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp515

    Analysis of nucleosomal array fibers. The pRST5 plasmid and expected fragments created by PvuII or SfaNI digestion ( A ). Micrococcal nuclease digestion at indicated time points of nucleosomal arrays reconstituted onto PvuII ( B ); SfaNI ( C ) digested pRST5 DNA. Multi-gels of telomeric nucleosomal array fibers (NA) and histone-free DNA (DNA) from pRST5 digested with PvuII ( D ); and SfaNI ( E ) prepared and subjected to electrophoresis according to ‘Materials and Methods’ section. Spheres refer to carboxylate-coated microsphere standards (35 nm radius). The ‘1 kb tel’ and ‘2 kb tel’ refer to the telomeric fragments liberated by PvuII and SfaNI digestion respectively. ‘N’ refers to the fragments without telomeric DNA. Logarithmic plot of pore sizes ( P e ) versus agarose% ( F ). Data was obtained from multi-gels run with bacteriophage T3 (Phage) in this laboratory or previous work [Phage, previous, ( 24 )] and 35 nm carboxylate-coated microspheres (Microspheres). P e for each agarose concentration was calculated according to ‘Materials and Methods’. Symbols with error bars represent the mean ± 1 SD of four to eight determinations.
    Figure Legend Snippet: Analysis of nucleosomal array fibers. The pRST5 plasmid and expected fragments created by PvuII or SfaNI digestion ( A ). Micrococcal nuclease digestion at indicated time points of nucleosomal arrays reconstituted onto PvuII ( B ); SfaNI ( C ) digested pRST5 DNA. Multi-gels of telomeric nucleosomal array fibers (NA) and histone-free DNA (DNA) from pRST5 digested with PvuII ( D ); and SfaNI ( E ) prepared and subjected to electrophoresis according to ‘Materials and Methods’ section. Spheres refer to carboxylate-coated microsphere standards (35 nm radius). The ‘1 kb tel’ and ‘2 kb tel’ refer to the telomeric fragments liberated by PvuII and SfaNI digestion respectively. ‘N’ refers to the fragments without telomeric DNA. Logarithmic plot of pore sizes ( P e ) versus agarose% ( F ). Data was obtained from multi-gels run with bacteriophage T3 (Phage) in this laboratory or previous work [Phage, previous, ( 24 )] and 35 nm carboxylate-coated microspheres (Microspheres). P e for each agarose concentration was calculated according to ‘Materials and Methods’. Symbols with error bars represent the mean ± 1 SD of four to eight determinations.

    Techniques Used: Plasmid Preparation, Electrophoresis, Concentration Assay

    TRF2 DBD-dependent changes in micrococcal nuclease digestion. Nucleosomal arrays (12.5 ng/ul) were incubated with indicated amounts of TRF2 DBD and digested with micrococcal nuclease according to ‘Materials and Methods’ section. Deproteinated samples were electrophoresed on a 12% native polyacrylamide gel and stained with SYBR Green ( A ) followed by transfer to a membrane and blotting with a biotin-d(TTAGGG) 7 probe ( B ) as described in the ‘Material and methods’ section. M refers to 100-bp ladder. The 1-, 2-, 3- to the left of the gel panels refer to mono-, di- and trinucleosomes, respectively.
    Figure Legend Snippet: TRF2 DBD-dependent changes in micrococcal nuclease digestion. Nucleosomal arrays (12.5 ng/ul) were incubated with indicated amounts of TRF2 DBD and digested with micrococcal nuclease according to ‘Materials and Methods’ section. Deproteinated samples were electrophoresed on a 12% native polyacrylamide gel and stained with SYBR Green ( A ) followed by transfer to a membrane and blotting with a biotin-d(TTAGGG) 7 probe ( B ) as described in the ‘Material and methods’ section. M refers to 100-bp ladder. The 1-, 2-, 3- to the left of the gel panels refer to mono-, di- and trinucleosomes, respectively.

    Techniques Used: Incubation, Staining, SYBR Green Assay

    Atomic force microscopy of TRF2 DBD-nucleosomal array complexes. A nucleosomal array fiber in the presence of 200 nM TRF2 DBD with a scale of the plane in the X and Y directions (0.43 and 0.43 μm, respectively) and height scale from 0 to 10 nm ( A ). Example of a height versus length plot of the complex in (A) obtained by Gwyddion software ( B ). Comparison of TRF2 DBD-dependent fiber height distributions by normalizing counts of fibers ( Supplementary Figure 4 ) having indicated heights to total number of fibers ( C ). Comparison of TRF2 DBD-dependent fiber contour length distributions by normalizing counts of fibers ( Supplementary Figure 5 ) having indicated contour lengths to total number of fibers ( D ). Contour lengths were generated with Chromatin Analysis 1.1.7 software.
    Figure Legend Snippet: Atomic force microscopy of TRF2 DBD-nucleosomal array complexes. A nucleosomal array fiber in the presence of 200 nM TRF2 DBD with a scale of the plane in the X and Y directions (0.43 and 0.43 μm, respectively) and height scale from 0 to 10 nm ( A ). Example of a height versus length plot of the complex in (A) obtained by Gwyddion software ( B ). Comparison of TRF2 DBD-dependent fiber height distributions by normalizing counts of fibers ( Supplementary Figure 4 ) having indicated heights to total number of fibers ( C ). Comparison of TRF2 DBD-dependent fiber contour length distributions by normalizing counts of fibers ( Supplementary Figure 5 ) having indicated contour lengths to total number of fibers ( D ). Contour lengths were generated with Chromatin Analysis 1.1.7 software.

    Techniques Used: Microscopy, Software, Generated

    TRF2 DBD-dependent changes in surface charge density ( μ ' 0 ), effective radius ( R e from dilute gels) and conformation flexibility ( R e versus P e ). The μ ' 0 at each indicated TRF2 DBD concentration was obtained by Ferguson plots of mobilities derived from multi-gels with agarose concentrations of 0.25–1% according to ‘Materials and Methods’ section ( A ). The R e at each TRF2 DBD concentration derived from 0.25–1% multi-gel experiments according to ‘Materials and Methods’ section ( B ). The R e values from 0.25–0.6% agarose (dilute gels) were averaged from each multi-gel experiment. The μ ' 0 or R e for each TRF2 DBD concentration was normalized to 0 nM TRF2 DBD. Each data point represents the mean ± 1 SD of 3-7 multi-gel experiments. The effect of the TRF2 DBD on the reptation of DNA and nucleosomal arrays (change in R e versus P e ) in concentrated gels ( C and D ). Plots of effective radius ( R e ) versus pore size ( P e ) of DNA ( C ) or nucleosomal arrays ( D ) incubated with indicated amounts of TRF2 DBD. R e and P e were derived from multi-gels of 0.7–2.5% agarose concentrations or 0.4–2.1% agarose concentrations according to ‘Materials and Methods’ section. The DNA fragment analyzed in all experiments in this figure was the 2-kb telomeric fragment unless otherwise indicated.
    Figure Legend Snippet: TRF2 DBD-dependent changes in surface charge density ( μ ' 0 ), effective radius ( R e from dilute gels) and conformation flexibility ( R e versus P e ). The μ ' 0 at each indicated TRF2 DBD concentration was obtained by Ferguson plots of mobilities derived from multi-gels with agarose concentrations of 0.25–1% according to ‘Materials and Methods’ section ( A ). The R e at each TRF2 DBD concentration derived from 0.25–1% multi-gel experiments according to ‘Materials and Methods’ section ( B ). The R e values from 0.25–0.6% agarose (dilute gels) were averaged from each multi-gel experiment. The μ ' 0 or R e for each TRF2 DBD concentration was normalized to 0 nM TRF2 DBD. Each data point represents the mean ± 1 SD of 3-7 multi-gel experiments. The effect of the TRF2 DBD on the reptation of DNA and nucleosomal arrays (change in R e versus P e ) in concentrated gels ( C and D ). Plots of effective radius ( R e ) versus pore size ( P e ) of DNA ( C ) or nucleosomal arrays ( D ) incubated with indicated amounts of TRF2 DBD. R e and P e were derived from multi-gels of 0.7–2.5% agarose concentrations or 0.4–2.1% agarose concentrations according to ‘Materials and Methods’ section. The DNA fragment analyzed in all experiments in this figure was the 2-kb telomeric fragment unless otherwise indicated.

    Techniques Used: Concentration Assay, Derivative Assay, Incubation

    TRF2 DBD binds specifically to telomeric DNA and nucleosomal arrays. 0.6% agarose gels of TRF2 DBD binding to DNA (DNA) ( A ) and nucleosomal arrays (NA) ( B ) from the pRST5 fragment digested to obtain a 1-kb DNA fragment with 580-bp telomeric DNA (telo) and 2.5-kb non-telomeric DNA (N-telo). Gels similar to (A) and (B), respectively except the pRST5 was digested to obtain a 2-kb fragment containing the 580-bp telomeric DNA (telo) with a 1 kb and smaller fragments being non-telomeric (N-telo) ( C and D ).
    Figure Legend Snippet: TRF2 DBD binds specifically to telomeric DNA and nucleosomal arrays. 0.6% agarose gels of TRF2 DBD binding to DNA (DNA) ( A ) and nucleosomal arrays (NA) ( B ) from the pRST5 fragment digested to obtain a 1-kb DNA fragment with 580-bp telomeric DNA (telo) and 2.5-kb non-telomeric DNA (N-telo). Gels similar to (A) and (B), respectively except the pRST5 was digested to obtain a 2-kb fragment containing the 580-bp telomeric DNA (telo) with a 1 kb and smaller fragments being non-telomeric (N-telo) ( C and D ).

    Techniques Used: Binding Assay

    The effect of full-length TRF2 and TRF2 DBD on the uptake of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/μl). Samples were incubated with indicated amounts of full-length TRF2 or TRF2 DBD and processed according to ‘Materials and Methods’. Uptake by nucleosomal arrays with samples incubated – or + 100 mM NaCl ( A ). The drawings on the side of the agarose gel refer to radiolabeled (*) T7 oligonucleotide either free or inserted into the nucleosomal arrays. A section of agarose gels showing T7 inserted into nucleosomal arrays ( top panel ), linear DNA ( middle panel ) and plasmid DNA ( bottom panel ) with increasing TRF2 DBD ( B ). Quantitation of (B) where uptake was normalized to 0 nM TRF2 DBD ( C ). Error bars are 1 SD of the mean of 3–5 determinations. Full-length TRF2-dependent uptake by nucleosomal arrays ( top panel ), linear DNA ( bottom panel ) ( D ). Quantitation of (D) where uptake was normalized to 0 nM TRF2 ( E ). The dashed line refers to uptake by supercoiled pRST5 from previous work ( 41 ). Error bars are 1 SD of the mean of three to four determinations.
    Figure Legend Snippet: The effect of full-length TRF2 and TRF2 DBD on the uptake of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/μl). Samples were incubated with indicated amounts of full-length TRF2 or TRF2 DBD and processed according to ‘Materials and Methods’. Uptake by nucleosomal arrays with samples incubated – or + 100 mM NaCl ( A ). The drawings on the side of the agarose gel refer to radiolabeled (*) T7 oligonucleotide either free or inserted into the nucleosomal arrays. A section of agarose gels showing T7 inserted into nucleosomal arrays ( top panel ), linear DNA ( middle panel ) and plasmid DNA ( bottom panel ) with increasing TRF2 DBD ( B ). Quantitation of (B) where uptake was normalized to 0 nM TRF2 DBD ( C ). Error bars are 1 SD of the mean of 3–5 determinations. Full-length TRF2-dependent uptake by nucleosomal arrays ( top panel ), linear DNA ( bottom panel ) ( D ). Quantitation of (D) where uptake was normalized to 0 nM TRF2 ( E ). The dashed line refers to uptake by supercoiled pRST5 from previous work ( 41 ). Error bars are 1 SD of the mean of three to four determinations.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Plasmid Preparation, Quantitation Assay

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    Worthington Biochemical nucleosomal arrays
    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of <t>nucleosomal</t> fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .
    Nucleosomal Arrays, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosomal arrays/product/Worthington Biochemical
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    nucleosomal arrays - by Bioz Stars, 2020-08
    88/100 stars
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    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Derivative Assay

    TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Centrifugation, Agarose Gel Electrophoresis

    The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Derivative Assay, Electrophoresis, Concentration Assay

    TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, Derivative Assay, Produced

    Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Microscopy

    The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis

    Analysis of nucleosomal array fibers. The pRST5 plasmid and expected fragments created by PvuII or SfaNI digestion ( A ). Micrococcal nuclease digestion at indicated time points of nucleosomal arrays reconstituted onto PvuII ( B ); SfaNI ( C ) digested pRST5 DNA. Multi-gels of telomeric nucleosomal array fibers (NA) and histone-free DNA (DNA) from pRST5 digested with PvuII ( D ); and SfaNI ( E ) prepared and subjected to electrophoresis according to ‘Materials and Methods’ section. Spheres refer to carboxylate-coated microsphere standards (35 nm radius). The ‘1 kb tel’ and ‘2 kb tel’ refer to the telomeric fragments liberated by PvuII and SfaNI digestion respectively. ‘N’ refers to the fragments without telomeric DNA. Logarithmic plot of pore sizes ( P e ) versus agarose% ( F ). Data was obtained from multi-gels run with bacteriophage T3 (Phage) in this laboratory or previous work [Phage, previous, ( 24 )] and 35 nm carboxylate-coated microspheres (Microspheres). P e for each agarose concentration was calculated according to ‘Materials and Methods’. Symbols with error bars represent the mean ± 1 SD of four to eight determinations.

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: Analysis of nucleosomal array fibers. The pRST5 plasmid and expected fragments created by PvuII or SfaNI digestion ( A ). Micrococcal nuclease digestion at indicated time points of nucleosomal arrays reconstituted onto PvuII ( B ); SfaNI ( C ) digested pRST5 DNA. Multi-gels of telomeric nucleosomal array fibers (NA) and histone-free DNA (DNA) from pRST5 digested with PvuII ( D ); and SfaNI ( E ) prepared and subjected to electrophoresis according to ‘Materials and Methods’ section. Spheres refer to carboxylate-coated microsphere standards (35 nm radius). The ‘1 kb tel’ and ‘2 kb tel’ refer to the telomeric fragments liberated by PvuII and SfaNI digestion respectively. ‘N’ refers to the fragments without telomeric DNA. Logarithmic plot of pore sizes ( P e ) versus agarose% ( F ). Data was obtained from multi-gels run with bacteriophage T3 (Phage) in this laboratory or previous work [Phage, previous, ( 24 )] and 35 nm carboxylate-coated microspheres (Microspheres). P e for each agarose concentration was calculated according to ‘Materials and Methods’. Symbols with error bars represent the mean ± 1 SD of four to eight determinations.

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Plasmid Preparation, Electrophoresis, Concentration Assay

    TRF2 DBD-dependent changes in micrococcal nuclease digestion. Nucleosomal arrays (12.5 ng/ul) were incubated with indicated amounts of TRF2 DBD and digested with micrococcal nuclease according to ‘Materials and Methods’ section. Deproteinated samples were electrophoresed on a 12% native polyacrylamide gel and stained with SYBR Green ( A ) followed by transfer to a membrane and blotting with a biotin-d(TTAGGG) 7 probe ( B ) as described in the ‘Material and methods’ section. M refers to 100-bp ladder. The 1-, 2-, 3- to the left of the gel panels refer to mono-, di- and trinucleosomes, respectively.

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: TRF2 DBD-dependent changes in micrococcal nuclease digestion. Nucleosomal arrays (12.5 ng/ul) were incubated with indicated amounts of TRF2 DBD and digested with micrococcal nuclease according to ‘Materials and Methods’ section. Deproteinated samples were electrophoresed on a 12% native polyacrylamide gel and stained with SYBR Green ( A ) followed by transfer to a membrane and blotting with a biotin-d(TTAGGG) 7 probe ( B ) as described in the ‘Material and methods’ section. M refers to 100-bp ladder. The 1-, 2-, 3- to the left of the gel panels refer to mono-, di- and trinucleosomes, respectively.

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Incubation, Staining, SYBR Green Assay

    Atomic force microscopy of TRF2 DBD-nucleosomal array complexes. A nucleosomal array fiber in the presence of 200 nM TRF2 DBD with a scale of the plane in the X and Y directions (0.43 and 0.43 μm, respectively) and height scale from 0 to 10 nm ( A ). Example of a height versus length plot of the complex in (A) obtained by Gwyddion software ( B ). Comparison of TRF2 DBD-dependent fiber height distributions by normalizing counts of fibers ( Supplementary Figure 4 ) having indicated heights to total number of fibers ( C ). Comparison of TRF2 DBD-dependent fiber contour length distributions by normalizing counts of fibers ( Supplementary Figure 5 ) having indicated contour lengths to total number of fibers ( D ). Contour lengths were generated with Chromatin Analysis 1.1.7 software.

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: Atomic force microscopy of TRF2 DBD-nucleosomal array complexes. A nucleosomal array fiber in the presence of 200 nM TRF2 DBD with a scale of the plane in the X and Y directions (0.43 and 0.43 μm, respectively) and height scale from 0 to 10 nm ( A ). Example of a height versus length plot of the complex in (A) obtained by Gwyddion software ( B ). Comparison of TRF2 DBD-dependent fiber height distributions by normalizing counts of fibers ( Supplementary Figure 4 ) having indicated heights to total number of fibers ( C ). Comparison of TRF2 DBD-dependent fiber contour length distributions by normalizing counts of fibers ( Supplementary Figure 5 ) having indicated contour lengths to total number of fibers ( D ). Contour lengths were generated with Chromatin Analysis 1.1.7 software.

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Microscopy, Software, Generated

    TRF2 DBD-dependent changes in surface charge density ( μ ' 0 ), effective radius ( R e from dilute gels) and conformation flexibility ( R e versus P e ). The μ ' 0 at each indicated TRF2 DBD concentration was obtained by Ferguson plots of mobilities derived from multi-gels with agarose concentrations of 0.25–1% according to ‘Materials and Methods’ section ( A ). The R e at each TRF2 DBD concentration derived from 0.25–1% multi-gel experiments according to ‘Materials and Methods’ section ( B ). The R e values from 0.25–0.6% agarose (dilute gels) were averaged from each multi-gel experiment. The μ ' 0 or R e for each TRF2 DBD concentration was normalized to 0 nM TRF2 DBD. Each data point represents the mean ± 1 SD of 3-7 multi-gel experiments. The effect of the TRF2 DBD on the reptation of DNA and nucleosomal arrays (change in R e versus P e ) in concentrated gels ( C and D ). Plots of effective radius ( R e ) versus pore size ( P e ) of DNA ( C ) or nucleosomal arrays ( D ) incubated with indicated amounts of TRF2 DBD. R e and P e were derived from multi-gels of 0.7–2.5% agarose concentrations or 0.4–2.1% agarose concentrations according to ‘Materials and Methods’ section. The DNA fragment analyzed in all experiments in this figure was the 2-kb telomeric fragment unless otherwise indicated.

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: TRF2 DBD-dependent changes in surface charge density ( μ ' 0 ), effective radius ( R e from dilute gels) and conformation flexibility ( R e versus P e ). The μ ' 0 at each indicated TRF2 DBD concentration was obtained by Ferguson plots of mobilities derived from multi-gels with agarose concentrations of 0.25–1% according to ‘Materials and Methods’ section ( A ). The R e at each TRF2 DBD concentration derived from 0.25–1% multi-gel experiments according to ‘Materials and Methods’ section ( B ). The R e values from 0.25–0.6% agarose (dilute gels) were averaged from each multi-gel experiment. The μ ' 0 or R e for each TRF2 DBD concentration was normalized to 0 nM TRF2 DBD. Each data point represents the mean ± 1 SD of 3-7 multi-gel experiments. The effect of the TRF2 DBD on the reptation of DNA and nucleosomal arrays (change in R e versus P e ) in concentrated gels ( C and D ). Plots of effective radius ( R e ) versus pore size ( P e ) of DNA ( C ) or nucleosomal arrays ( D ) incubated with indicated amounts of TRF2 DBD. R e and P e were derived from multi-gels of 0.7–2.5% agarose concentrations or 0.4–2.1% agarose concentrations according to ‘Materials and Methods’ section. The DNA fragment analyzed in all experiments in this figure was the 2-kb telomeric fragment unless otherwise indicated.

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Concentration Assay, Derivative Assay, Incubation

    TRF2 DBD binds specifically to telomeric DNA and nucleosomal arrays. 0.6% agarose gels of TRF2 DBD binding to DNA (DNA) ( A ) and nucleosomal arrays (NA) ( B ) from the pRST5 fragment digested to obtain a 1-kb DNA fragment with 580-bp telomeric DNA (telo) and 2.5-kb non-telomeric DNA (N-telo). Gels similar to (A) and (B), respectively except the pRST5 was digested to obtain a 2-kb fragment containing the 580-bp telomeric DNA (telo) with a 1 kb and smaller fragments being non-telomeric (N-telo) ( C and D ).

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: TRF2 DBD binds specifically to telomeric DNA and nucleosomal arrays. 0.6% agarose gels of TRF2 DBD binding to DNA (DNA) ( A ) and nucleosomal arrays (NA) ( B ) from the pRST5 fragment digested to obtain a 1-kb DNA fragment with 580-bp telomeric DNA (telo) and 2.5-kb non-telomeric DNA (N-telo). Gels similar to (A) and (B), respectively except the pRST5 was digested to obtain a 2-kb fragment containing the 580-bp telomeric DNA (telo) with a 1 kb and smaller fragments being non-telomeric (N-telo) ( C and D ).

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Binding Assay

    The effect of full-length TRF2 and TRF2 DBD on the uptake of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/μl). Samples were incubated with indicated amounts of full-length TRF2 or TRF2 DBD and processed according to ‘Materials and Methods’. Uptake by nucleosomal arrays with samples incubated – or + 100 mM NaCl ( A ). The drawings on the side of the agarose gel refer to radiolabeled (*) T7 oligonucleotide either free or inserted into the nucleosomal arrays. A section of agarose gels showing T7 inserted into nucleosomal arrays ( top panel ), linear DNA ( middle panel ) and plasmid DNA ( bottom panel ) with increasing TRF2 DBD ( B ). Quantitation of (B) where uptake was normalized to 0 nM TRF2 DBD ( C ). Error bars are 1 SD of the mean of 3–5 determinations. Full-length TRF2-dependent uptake by nucleosomal arrays ( top panel ), linear DNA ( bottom panel ) ( D ). Quantitation of (D) where uptake was normalized to 0 nM TRF2 ( E ). The dashed line refers to uptake by supercoiled pRST5 from previous work ( 41 ). Error bars are 1 SD of the mean of three to four determinations.

    Journal: Nucleic Acids Research

    Article Title: The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

    doi: 10.1093/nar/gkp515

    Figure Lengend Snippet: The effect of full-length TRF2 and TRF2 DBD on the uptake of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/μl). Samples were incubated with indicated amounts of full-length TRF2 or TRF2 DBD and processed according to ‘Materials and Methods’. Uptake by nucleosomal arrays with samples incubated – or + 100 mM NaCl ( A ). The drawings on the side of the agarose gel refer to radiolabeled (*) T7 oligonucleotide either free or inserted into the nucleosomal arrays. A section of agarose gels showing T7 inserted into nucleosomal arrays ( top panel ), linear DNA ( middle panel ) and plasmid DNA ( bottom panel ) with increasing TRF2 DBD ( B ). Quantitation of (B) where uptake was normalized to 0 nM TRF2 DBD ( C ). Error bars are 1 SD of the mean of 3–5 determinations. Full-length TRF2-dependent uptake by nucleosomal arrays ( top panel ), linear DNA ( bottom panel ) ( D ). Quantitation of (D) where uptake was normalized to 0 nM TRF2 ( E ). The dashed line refers to uptake by supercoiled pRST5 from previous work ( 41 ). Error bars are 1 SD of the mean of three to four determinations.

    Article Snippet: Micrococcal nuclease digestion To validate proper reconstitution, an aliquot of 0.5 μg of reconstituted nucleosomal arrays was digested for indicated times ( B and C) with 12 units of micrococcal nuclease (Worthington) in reaction buffer containing 20 mM Tris–HCl and 2 mM CaCl2 (final concentrations) in a total of 20 μl.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Plasmid Preparation, Quantitation Assay