protease inhibitor cocktail  (thermo fisher)

Journal: Nature Communications

Article Title: Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

doi: 10.1038/s41467-017-00599-0

Figure Lengend Snippet: Peptidase activity assays of purified recombinant FliA(H)-hypervariable region and flagellar filaments from C. haemolyticum . a 12% SDS-PAGE of purified FliA(H)-hypervariable region (band “i”), electrophoresing close to that predicted for recombinant thioredoxin-tagged flagellin hypervariable region (50.14 kDa). b Amino acid occurrence heat map based on FliA(H)-hypervariable region protease cleaved peptide alignments of 391 cleavage sites identified in a tryptic E. coli K12 library. c IceLogos of FliA(H)-hypervariable region protease cleaved peptide specificity profiles showing percent differences compared to natural amino acid abundance, with significantly over-represented amino acids shown above the x -axis and under-represented residues below the x -axis. Amino acids that have not been identified are depicted in pink . d Fluorometric peptidase assays for FliA(H)-hypervariable region protease against three different peptidic substrates (ALG↓L, PLG↓L, PLG↓V) and + /– EDTA as indicated. e 10% SDS-PAGE of purified flagellar sheared filaments from C. haemolyticum . Band “ii” and “iii” were identified by mass spectrometry as FliA(H) (proteolytic flagellin) and the non-protease-containing structural flagellin (WP_039229459), respectively (Supplementary Fig. 7 ). f Amino acid occurrence heat map and g IceLogo based on cleaved peptide alignments of 269 cleavage sites identified in a tryptic E. coli K12 library using purified flagellar filaments. h Fluorometric peptidase assays for flagellar filaments using the ALG↓L peptide in three assay conditions including EDTA-free HALT protease inhibitors

Article Snippet: Purified recombinant FliA(H)-hypervariable region protease was incubated at a final concentration of 0.5 µM with 10 µM QF-peptide substrate in 100 µl of 150 mM NaCl, 10 mM CaCl2 , 50 mM HEPES, pH 7.5, in the presence of HALT protease inhibitor cocktail (Life Technologies) plus/minus 20 mM EDTA at 37 °C.

Techniques: Activity Assay, Purification, Recombinant, SDS Page, Mass Spectrometry

Journal: Gene therapy

Article Title: Recombinant elastin based nanoparticles for targeted gene therapy

doi: 10.1038/gt.2017.54

Figure Lengend Snippet: Lentiviral vector is released from LDLR3-ELP after internalization by cells A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.

Article Snippet: The cells were then lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA) (Thermo scientific cat #78440).

Techniques: Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: bioRxiv

Article Title: Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

doi: 10.1101/2020.09.09.289306

Figure Lengend Snippet: N-Cadherin from POMGNT1-deficient cells shows enhanced homotypic interactions in vitro . (A) Semi-quantitative analysis of the mean size of dynabead aggregates coated with the recombinant extracellular domain of recombinant N-Cdh purified from WT (gray line) and ΔPOMGnT1 cells (black line). Protein-coated dynabeads were imaged following incubation in the presence of either CaCl 2 (solid lines) or EDTA (broken lines) at the indicated time intervals. Mean aggregate size is plotted against time. Assays were performed at quadruplicate from two independent experiments. Data are represented as means ± SD. Asterisks denote statistical significance in comparison to WT cells: * p ≤ 0.05, ** p ≤ 0.01. (B) Western Blot analysis evaluating levels of protein from WT and ΔPOMGnT1 (Δ) bound to dynabeads. Dynabead-bound recombinant N-Cdh was eluted in SDS sample buffer (beads), resolved on a 10% SDS PAA gel. For detection on Western blot an anti-5xHis antibody was used. In addition, protein before binding to beads (input) and unbound fractions (void) are shown.

Article Snippet: Whole cell lysate and crude membrane preparation Cells at 90% confluency (~5.0 x 106 cells) were washed with ice-cold PBS and lysed with 500 μl of lysis buffer (10 mM Tris-HCl pH 7.6, 1% (w/v) SDS, 1 mM EDTA, supplemented with Halt™ protease and phosphatase inhibitor cocktail without EDTA (ThermoFisher Scientific)).

Techniques: In Vitro, Recombinant, Purification, Incubation, Western Blot, Binding Assay

Journal: Journal of Tissue Engineering

Article Title: Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis

doi: 10.1177/2041731414566529

Figure Lengend Snippet: Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ ™ software and relative protein expression levels were computed with respect to β-actin.

Article Snippet: Protein isolation and western blotting analysis At the end of 14 days of culture, cells were released from scaffolds with 1X Trypsin followed by pelleting cells at 1000×g and finally lysing the pellet with Pierce IP lysis buffer supplemented with 1X Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA).

Techniques: Expressing, Lysis, SDS Page, Western Blot, Incubation, Software