bsa  (New England Biolabs)

Journal: Journal of Bacteriology

Article Title: The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

doi: 10.1128/JB.00653-17

Figure Lengend Snippet: Electrophoresis mobility shift assay (EMSA) and DNA footprinting assays indicate HlyU binding to a region within the exsA promoter. (A) Six percent TBE-polyacrylamide gels were loaded with reaction mixtures containing exsA promoter DNA and increasing amounts of purified HlyU-His or bovine serum albumin (BSA). An exsA promoter DNA-only control was included as indicated. The white box indicates slow-migrating, concentrated DNA species. (B) A 6% TBE-polyacrylamide gel was loaded with reaction mixtures containing nleH1 DNA and increasing amounts of purified HlyU-His. Following electrophoresis, gels were stained with SYBR green to specifically stain DNA. (C) A DNase I footprinting assay using purified HlyU-His was used to identify a DNA binding region for HlyU-His within the exsA promoter. The approximate base pair numbers of the HlyU-His footprint region are indicated (259 to 315 bp) and are based on capillary electrophoresis internal size standards. The DNA sequence associated with the identified HlyU protected region is displayed below the chromatogram. A 7-bp inverted repeat (labeled in blue) and separated by 14 bp is centrally located within the HlyU-His protected region.

Article Snippet: Purified HlyU-His or BSA (B9000S; New England BioLabs) was mixed with a PCR-amplified exsA promoter DNA fragment in binding buffer (10 mM Tris [pH 7.5 at 20°C], 1 mM EDTA, 0.1 M KCl, 0.1 mM dithiothreitol, 5%, vol/vol, glycerol, 0.01 mg ml−1 BSA).

Techniques: Electrophoresis, Mobility Shift, DNA Footprinting, Binding Assay, Purification, Staining, SYBR Green Assay, Footprinting, Sequencing, Labeling

Journal: PLoS ONE

Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform

doi: 10.1371/journal.pone.0225081

Figure Lengend Snippet: Enzymatic properties of recombinant hENDOV proteins. (A) RNA cleavage activity of hENDOV 282 (282), hENDOV 308 (308) and hENDOV 309 (309) enzymes. Increasing amounts of enzyme (20, 40, 80 and 160 fmol) were incubated with a single stranded (ss; upper) or double-stranded (ds; lower) RNA substrates containing inosine and reaction products were analyzed by denaturing gel electrophoresis. Quantification of the cleavage activity by ImageQuant TL software (n = 3) is shown below. (B) Electrophoretic mobility shift assay using hENDOV 282 (282), hENDOV 308 (308) and hENDOV 309 (309) enzymes (1, 2 or 3 pmol) and ss (left panel) or ds (right panel) RNA substrates containing inosine. Reactions were incubated on ice prior to separation of enzyme-substrate complexes from unbound substrate on native polyacrylamide gels. Quantification of band shifts by ImageQuant TL software (n = 3) is shown below. (C) Representative images of northern blot analyses of total RNA from HAP1 cells (WT) incubated with recombinant hENDOV 282 (282), hENDOV 308 (308) and hENDOV 309 (309) enzymes (75 and 150 pmol). The probes used recognize AlaAGC5’, ArgACG5’ or ValAAC5’ tRNAs. Sample without enzyme (-) or with 150 pmol BSA were included as controls. Equal loading is shown by ethidium bromide (Eth. Br.) staining of the gel (lower panel). Glyphs to the right of the membranes indicate full-length and fragmented tRNA species.

Article Snippet: A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

Techniques: Recombinant, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Software, Electrophoretic Mobility Shift Assay, Northern Blot, Staining