Journal: Scientific Reports

Article Title: Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis

doi: 10.1038/s41598-020-66857-2

Figure Lengend Snippet: Agarose gel electrophoresis of DNA extracts from BCC9546. ( a ) Genomic DNA running on 1% agarose gel: M, 250 ng DNA size marker (GeneRuler DNA Ladder Mix, Thermo Scientific); 1–2, 250 ng BCC9546 genomic DNA extract. ( b ) Plasmid DNA running on 0.5% agarose gel: Ms, 250 ng supercoiled DNA Ladder (New England BioLabs); p1, 50 ng BCC9546 plasmid DNA extract; p2, 120 ng BCC9546 plasmid DNA extract. The gel images were cropped for concise visualization. See Supplementary Fig. for the uncropped gel images.

Article Snippet: Figure 2 Agarose gel electrophoresis of DNA extracts from BCC9546. ( a ) Genomic DNA running on 1% agarose gel: M, 250 ng DNA size marker (GeneRuler DNA Ladder Mix, Thermo Scientific); 1–2, 250 ng BCC9546 genomic DNA extract. ( b ) Plasmid DNA running on 0.5% agarose gel: Ms, 250 ng supercoiled DNA Ladder (New England BioLabs); p1, 50 ng BCC9546 plasmid DNA extract; p2, 120 ng BCC9546 plasmid DNA extract.

Techniques: Agarose Gel Electrophoresis, Marker, Plasmid Preparation

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: RAD52 stimulates RAD51 mediated dsDNA unwinding . Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 (3.0 μM) in presence of increasing concentration of RAD52 (0 μM, 0.5 μM, 1.0 μM & 2.0 μM) (lanes 1–4 in each Panel) in binding buffer R at 37°C for 12 minutes, in presence of either ATP, ADP, AMP-PNP, ATPγS, or AMP (1 mM each) (Panels A-E) or no nucleotide (Panel F), followed by deproteinization and agarose gel assay (Methods). Lane 5 in all the Panels has all the components except RAD51. Panel G represents only RAD52 control in presence of ATP. Panel H represents no protein control. Percentage unwound DNA (normalized and quantified) was expressed as a ratio of the intensity associated with unwound DNA band to the sum of intensities corresponding to unwound as well as relaxed dsDNA bands. This was plotted as a function of RAD52 concentration (Panel I). Data points were statistically analyzed as explained earlier.

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Incubation, Concentration Assay, Binding Assay, Agarose Gel Electrophoresis

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: RAD52 stimulates RAD51 mediated dsDNA unwinding even in presence of ADP: Time-course analysis . Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 (3.0 μM) in presence of RAD52 (2.0 μM) in binding buffer R at 37°C with either 1 mM ATP (Panel A) or ADP (Panel B). Reactions were terminated after indicated time points by SDS/Proteinase K treatment and analyzed on agarose gels. Lane 6 in both Panels represents only RAD52 control while lane 7 represents only RAD51 control. Percentage intensity of unwound DNA to total DNA per lane (as explained earlier) was plotted as a function of time (Panel C).

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Incubation, Binding Assay

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: RAD52 promotes formation of large complexes of RAD52-RAD51-DNA: gel shift assay . Supercoiled φX174 DNA (30 μM) was incubated with increasing concentrations of RAD51 (3 μM, 6 μM & 9 μM) either in presence or absence of RAD52 (2 μM) in binding buffer R at 37°C for 12 minutes, in presence of either ATP, ADP, ATPγS, AMP-PNP, or AMP (1 mM each) or no nucleotide, followed by agarose gel assay (Methods). Controls (Lane 1 in Panel A: no proteins; Panel D: no RAD51). Lane 5 in Panel A was excised (from the region shown in thatched box) and subjected to analyses on 12% SDS-PAGE gel followed by Coomassie staining (lane 1, Panel E). Purified RAD51 (lane 2, Panel E) and RAD52 proteins (lanes 3, Panel E).

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Agarose Gel Electrophoresis, SDS Page, Staining, Purification

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: RAD52 promotes coaggregation of RAD52-RAD51-DNA complexes: centrifugation assay followed by protein analyses . Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 (3.0 μM) in presence of increasing concentration of RAD52 (0 μM, 0.5 μM, 1.0 μM & 2.0 μM) in binding buffer R at 37°C for 12 minutes, in presence of either ATP, ADP, AMP-PNP, ATPγS, or AMP (1 mM each) (Panels E-I) or no nucleotide (Panel J), followed by centrifugation assay (Methods) and analyses of proteins by SDS-PAGE (Methods). Lanes s1–s4 and p1–p4 in all the Panels represent supernatant and pellet fractions for increasing concentrations of RAD52 respectively. Lanes 1 & 2 of Panel A show input RAD52 and RAD51 respectively used in centrifugation assay. No DNA controls: lanes s1 and p1 of Panel C correspond to supernatant and pellet fractions for RAD51 (3 μM) and RAD52 (2 μM) together, while the same for Panels B and D correspond to RAD52 protein alone and RAD51 protein alone, respectively. Intensity of protein bands was quantified using Image J software. Percentage of RAD51 protein in pellet was expressed as a ratio of the total intensity associated with pellet and supernatant protein bands and plotted as a function of RAD52 protein concentration (Panel K) (percentage of protein in supernatant fraction will therefore be 100% minus pellet fraction). The quantitative data has been given for RAD52 corresponding to only ATP set.

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Centrifugation, Incubation, Concentration Assay, Binding Assay, SDS Page, Software, Protein Concentration

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: KCl induced changes in the aggregation of RAD51-dsDNA complexes: protein and DNA analyses . Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 (3.0 μM) in presence of increasing concentration of KCl [0 mM, 25 mM, 50 mM, 100 mM, 140 mM, & 200 mM] with 1 mM ATP either in presence of RAD52 [2.0 μM] (Panel B: Protein analysis, Panel D:DNA analysis) or in its absence (Panel A: Protein analysis, Panel C:DNA analysis) in binding buffer R at 37°C for 12 minutes followed by centrifugation assay (Methods) and analyses of proteins by SDS-PAGE (Methods). Lanes s1–s6 and p1–p6 in all the Panels represent supernatant and pellet fractions for increasing concentrations of KCl respectively. RAD51 bands in pellet fraction is expressed as a fraction of the protein band intensities associated with the sum of supernatant and pellet fractions and plotted as a function of KCl concentration [(Open square: Panel E) in presence of RAD52] [(Open diamond: Panel E) in absence of RAD52)]. Similarly RAD52 protein is also represented in Panel E (open triangle). For DNA analysis, samples were deproteinized (Methods). Percentage of DNA (relaxed and unwound forms) in pellet fraction was expressed as a ratio of the total DNA (relaxed and unwound forms) in both pellet and supernatant fractions and plotted as a function of KCl concentration [(Filled square: Panel E) presence of RAD52 & (Filled diamond: Panel E) absence of RAD52].

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Incubation, Concentration Assay, Binding Assay, Centrifugation, SDS Page

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: KCl induced changes in dsDNA unwinding by RAD51 . Relaxed φX174 DNA (30 μM) along with Topo I was incubated with RAD51 [3.0 μM] in presence of increasing concentration of KCl [0 mM, 10 mM, 25 mM, 50 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM & 200 mM] with ATP either in presence of RAD52 [2.0 μM] (Panel B: lanes 1–10) or in its absence (Panel A: lanes 2–11). Lane 1 in Panel A represents no protein control. Unwound DNA band intensity was expressed as percentage of total DNA band intensities per lane and plotted as a function of KCl concentration (Panel C).

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Incubation, Concentration Assay

Journal: BMC Biochemistry

Article Title: Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

doi: 10.1186/1471-2091-10-2

Figure Lengend Snippet: Effect of KCl on RAD51 binding to dsDNA: gel shift assay . Supercoiled φX174 DNA (30 μM) was incubated with RAD51 (3 μM) with ATP (1 mM) in binding buffer R at 37°C for 12 minutes in presence of increasing concentrations of KCl [0 mM, 25 mM, 50 mM, 100 mM, 140 mM & 200 mM] (lanes 2–7), followed by gel-shift analyses (Methods). Lane 1 represents no protein control.

Article Snippet: φX174 supercoiled DNA substrate was obtained from New England Biolabs.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation