Journal: mBio

Article Title: Preferential Packing of Acidic Glycosidases and Proteases into Bacteroides Outer Membrane Vesicles

doi: 10.1128/mBio.00909-14

Figure Lengend Snippet: OMV display protease activity. (A) Ten micrograms of OMV proteins was loaded on 10% Tris-glycine gel with 0.1% gelatin as the substrate. Following separation at denaturing conditions, proteins were renatured and then incubated at 37°C for 2 days to allow substrate cleavage. Colloidal Coomassie brilliant blue G-250 was used to stain the gel. Clear bands marked by the arrows indicate the digestion of gelatin by proteases. (B) Peptidase activity of B. fragilis vesicles was tested using different p -nitroanilide-linked amino acids. Ten micrograms of purified OMV proteins was incubated with different substrates for 1 h at 37°C. Activities were determined by measuring the absorbance of the released p -nitroanilide at 405 nm. All activities are represented relative to the alanine-peptidase activity. All experiments were done in triplicates.

Article Snippet: The peptidase enzyme activity was determined by incubation of 10 µg of the quantified OMV proteins in a 50 mM Tris-HCl buffer (pH 6.5), with 200 µl of 50 mM l -lysine-p -nitroanilide dihydrobromide, l -alanine-p -nitroanilide hydrochloride, l -leucine-p-nitroanilide, or pyroglutamic acid-p -nitroanilide (Sigma-Aldrich), for 1 h at 37°C.

Techniques: Activity Assay, Incubation, Staining, Purification

Journal: Journal of Bacteriology

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿

doi: 10.1128/JB.00443-10

Figure Lengend Snippet: Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.

Article Snippet: Protein purification was verified by electrophoresis using a 4 to 15% Tris-HCl, SDS-polyacrylamide gel (Bio-Rad) and visualized by staining with Imperial protein stain (Bio-Rad).

Techniques: Recombinant, In Vitro, Clone Assay, Purification, Staining, Western Blot, Mass Spectrometry, Paper Chromatography, Incubation, Negative Control, Modification

Journal: Sensors (Basel, Switzerland)

Article Title: Combining Electrochemical Impedance Spectroscopy and Surface Plasmon Resonance into one Simultaneous Read-Out System for the Detection of Surface Interactions

doi: 10.3390/s131114650

Figure Lengend Snippet: Alternating the media 10 mM Tris-HCl and 1× PBS buffer for three consecutive runs. ( A ) Shows the minimum shift when exchanging the media. ( B ) Illustrates the changes in amplitude and its phase at a frequency of 251 Hz, when exchanging the media. Comparing the signals, when plotted in the same graph, reveals that the read-out systems detected the media exchange simultaneously, without influencing each other ( C ).

Article Snippet: Proof of Principle To assess the working principle of the combined unit, the reaction chamber of the PDMS flow-cell was filled sequentially with 10 mM Tris-HCl (Carl Roth, Karlsruhe, Germany) and 1× PBS buffer (pH 7.4; GIBCO Life Technologies GmbH, Darmstadt, Germany).

Techniques:

Journal: Bioinorganic Chemistry and Applications

Article Title: DNA Interaction and DNA Cleavage Studies of a New Platinum(II) Complex Containing Aliphatic and Aromatic Dinitrogen Ligands

doi: 10.1155/2011/525794

Figure Lengend Snippet: Cleavage of SC pUC19 DNA (50 μ M) by platinum complex (100 μ M) in the presence of H 2 O 2 (100 μ M), in 10 mM Tris-HCl/1 mM EDTA buffer (pH 8.0). lane 1: DNA Marker; lane 2: DNA control; lane 3: DNA + complex; lane 4: DNA + complex 1 + H 2 O 2 ; lane 5: DNA + complex + H 2 O 2 + DMSO (4 μ L); lane 6: DNA + complex 1 + H 2 O 2 + NaN 3 (100 μ M).

Article Snippet: Materials 4,7-diphenyl-1,10-phenanthroline (DIP), N,N -dimethyltrimethylenediamine, hydrazine dihydrochloride, potassium Chloride, nitric acid, Tris-acetate-EDTA (TAE) buffer, hydrogen peroxide, NaN3 , and Tris-HCl were purchased from Merck.

Techniques: Marker

Journal: Metallomics : integrated biometal science

Article Title: Human Calprotectin Affects the Redox Speciation of Iron

doi: 10.1039/c7mt00044h

Figure Lengend Snippet: Iron depletion by CP-Ser, ΔHis 3 Asp and ΔHis 4 under aerobic conditions. (A) Cartoon schematic of the metal-depletion assay. Bacterial growth medium was treated with 10.5 μM CP (250 μg/mL) and incubated at 30 ºC, 150 rpm. At a given time point, an aliquot of medium was transferred to a spin filter. Following centrifugation to remove CP, and the CP-treated medium was analyzed by ICP-MS. (B–D) Tris:TSB treated CP-Ser, ΔHis 3 Asp, or ΔHis 4 in the absence (black) or presence (red) of ≈3 mM BME. The metal content in the CP-treated medium was analyzed by ICP-MS at t = 0, 1, 2, 4, 8, 24, and 48 h. The mean and SDM are reported ( n .

Article Snippet: Tris buffer (50 mL) containing 20 mM Tris, 100 mM NaCl, 2 mM CaCl2 , pH 7.5 in the absence and presence of 5 mM BME (Alfa Aesar) was prepared by diluting stock solutions of 1.0 M Tris-HCl (molecular biology grade, Alfa Aesar), 1.0 M NaCl (molecular biology grade, Sigma), and 1.0 M CaCl2 .

Techniques: Depletion Assay, Incubation, Centrifugation, Mass Spectrometry

Journal: The EMBO Journal

Article Title: Direct association of ligand-binding and pore domains in homo- and heterotetrameric inositol 1,4,5-trisphosphate receptors

doi: 10.1093/emboj/19.20.5450

Figure Lengend Snippet: Fig. 4. Cross-linking of N- and C-termini by DTSSP. ( A ) Co-transfected microsomes were subjected to trypsin digestion (lanes 2, 3 and 5–9) or mock digested (lanes 1 and 4) and pelleted. Microsomes in lane 3 and lanes 6–9 were then treated with 0.5 mM DTSSP for 30 min and the reaction was terminated by the addition of 20 mM Tris pH 7.5 as described in Materials and methods. All samples were then solubilized by the addition of 1% Zwittergent, immunoprecipitated with CT1 and subjected to SDS–PAGE. Lanes 1–3 were probed with an anti-Myc antibody and the same immunoblot was stripped and re-probed with a FLAG antibody (lanes 4–6). Cross-linked N-terminal peptides co-precipitating with CT1 are designated by a closed (type I) or open (type III) circle. No DTSSP-specific bands in trypsin treated samples were observed when the same immunoblot was probed with antibodies raised against amino acids 401–414 (KEEK, lane 7) and 1883–1902 (ABR, lane 8) of the type I receptor. Similarly, no immunoreactive bands were observed when the immunoblot was probed with an antibody to the type III C-terminus (CT3, lane 9). ( B ) Samples were treated as in (A), except that increasing concentrations (0.5–5.0 mM) of cross-linker were used in lanes 2–6 and lanes 8–12. Increasing the DTSSP concentration was accompanied by a shift in mobility of the 42 kDa N-terminal fragment in a dose-dependent manner (indicated by arrows, see text for details). As in (A), when the blot in (B) was stripped and reprobed with KEEK, ABR or CT3 antibodies, no immunoreactive reactive bands were observed in trypsin-treated samples (data not shown). *IgG band.

Article Snippet: Pellets were either quenched with SDS–PAGE sample buffer directly or resuspended in 150 mM NaCl, 50 mM Tris–HCl pH 7.8, 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF and 1× complete protease inhibitor cocktail (Roche Molecular Biochemicals) for immunoprecipitation assays.

Techniques: Transfection, Immunoprecipitation, SDS Page, Concentration Assay