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: mBio

Article Title: TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response

doi: 10.1128/mbio.00633-22

Figure Lengend Snippet: Conserved cysteine residue (C24) and its role in OxsR function. (A) Impact of OxsR C24A on the relative-fold change of hvo_1043 transcript levels during hypochlorite stress. H. volcanii strains as indicated on the x axis were grown to early exponential phase in GMM and treated with 0 and 2.5 mM NaOCl for 15 min. This time frame was found to result in a 10- to 40-fold increased abundance of hvo_1043 transcripts in the parent strain. Total RNA was extracted and used for qRT-PCR analysis. The internal reference hvo_1015 normalized levels of the gene expression were at 1-fold relative fold change. Significant differences between the parent and mutant by the Student's t test analysis (**, P -value ≤ 0.001; *, P -value ≤ 0.05). n.s., not significant. (Exp./Bio:2 to 4; Tech: 3 replicates). (B) Detection of OxsR-HA with and without the C24A variant in H. volcanii cells. Strains were grown to early log phase (OD600 of 0.3 to 0.5). Upper panel: Immunoprecipitates (10 μL per lane) were separated by reducing 15% SDS-PAGE for 2 h at 100 V. Proteins were detected by immunoblotting analysis using anti-HA tag HRP (# ab1190) antibodies at 1:20,000 dilution and ECL Prime. Signal was visualized after a 30-s exposure. Molecular mass standards were Precision Plus Protein Kaleidoscope. Lower panel: Total protein input separated by reducing 12% SDS-PAGE prior to immunoprecipitation detected by Sypro Ruby staining is included as control. Samples were normalized as 4 μL (0.04 OD 600 units cell pellet) per lane. (C) OxsR forms an intersubunit disulfide bond. OxsR wt and C24A proteins were purified with a C-terminal StrepII tag and separated by reducing and nonreducing 16% SDS-PAGE as indicated. (D) Analysis of OxsR binding to DNA by EMSA. Reactions (12 μL) were separated by 9% PAGE in 0.5×TBE buffer pH 8.3 after incubation with 1% formaldehyde for 10 min at room temperature in 50 mM HEPES, pH 7.5, 2 M NaCl, 10% glycerol, 15 mM MgCl 2 , 1 mM EDTA supplemented with 0.25 μg/μL BSA and 0.1 μg/μL sheered salmon sperm DNA. Biotinylated (*) probe was added as indicated. OxsR wt and C24A protein concentrations were based on purified homodimer. See Materials and Methods for details.

Article Snippet: These OxsR proteins (34 nM to 5 μM) were incubated (12 μL reactions) with the 5′-biotinylated dsDNA PCR products (0.5 to 2.5 nM) in binding buffer supplemented with ultrapure sheared salmon sperm DNA at 0.1 μg/mL (Ambion AM9680) and molecular biology grade BSA at 0.25 μg/mL (New England Biolabs) for 10 min at room temperature with 1% (vol/vol) formaldehyde.

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Variant Assay, SDS Page, Immunoprecipitation, Staining, Purification, Binding Assay, Polyacrylamide Gel Electrophoresis, Incubation

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