Cells were lysed in 1% NP-40 lysis buffer (150 mM NaCl, 20 mM HEPES, 0.5 mM EDTA, 1 mM Na3VO4, proteinase inhibitor cocktail (Calbiochem). Total cell extracts concentration was determined using Coomassie Brilliant Blue staining (Sigma-Aldrich) and resolved in 10% acrylamide gels and transferred onto PVDF membrane (Millipore, MA, USA) before immunoblotting with the appropriate antibodies overnight at 4 °C. Primary antibody incubation was followed by incubation with an HRP-conjugated secondary antibody (anti-goat, anti-rabbit; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA and anti-mouse; CST, Danvers, MA, USA).
Coomassie brilliant blue staining
Manufactured by Merck Group
Sourced in United States
Coomassie Brilliant Blue is a laboratory dye used for the staining of proteins in gel electrophoresis. It binds non-specifically to proteins, allowing for the visualization and quantification of protein bands in polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Protein a g sepharose, by Abcam (1 mentions)
Dna extraction kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Clc main workbench 5, by Qiagen (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
5 protocols using coomassie brilliant blue staining
1
Cell Lysis and Immunoblotting Protocol
2
Purification of Anti-HS scFv Antibodies
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VSV-tagged anti-HS single chain (scFv) antibodies (Table 1 ) AO4B08, EW3D10, EW4G2, defining inflammatory HS domains [16 (link), 24 (link), 25 (link)] and a control anti-HS scFv antibody HS4C3, which detects a non-inflammatory HS domain [23 (link)], were produced in E.coli. Antibodies were purified by cobalt resin affinity purification (Life Technologies, Breda, The Netherlands), followed by characterization of HS binding affinity in ELISA with coated heparan sulfate from bovine kidney (HSBK, Sigma-Aldrich, Zwijndrecht, The Netherlands) and analysis of the purity via SDS-PAGE (Bio-Rad, Veenendaal, The Netherlands) and by Western blotting (Bio-Rad) or coomassie brilliant blue staining (Sigma-Aldrich, Zwijndrecht, The Netherlands) as described previously [28 (link)]. Antibodies were concentrated in 10 kDa Amicon centrifuge concentration tubes (Merck chemicals B.V., Amsterdam, The Netherlands).
3
Immunoprecipitation and Mass Spectrometry Analysis
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Total proteins were extracted using Mammalian Protein Extraction Reagent (Thermo) supplemented with a complete protease inhibitor cocktail (Roche). Immunoprecipitations were performed by incubating whole cell extracts with the indicated antibody for 6 h at 4 °C with rocking after a preincubation with Protein A/G-Sepharose (Abcam). Immunoprecipitates were washed and resolved by SDS-PAGE. For MS, after Coomassie Brilliant blue staining (Sigma-Aldrich), total protein bands in the lane were excised and analysed by ion-trap MS at Shanghai Genechem Co., Ltd. Antibodies against Flag (No. 8146), Myc (No. 2272), anti-HA (No. 3724), SOCS1 (No. 3950), STAT3 (No. 9139), pSTAT3 (No. 9131), JAK1 (No. 3332), pJAK1 (No. 3331) and Ub (No. 3936) were purchased from Cell Signalling Technology.
4
Identification and Characterization of AHL-Degrading Bacteria
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Identification of the bacterial isolate possessing the most significant degradation percentage against different AHLs signals was done by 16S rRNA gene amplification and sequencing. Genomic DNA of the isolate was extracted using a DNA extraction kit (Qiagen). The 16S rRNA gene was amplified by PCR using universal primers 27F: (5‘-AGA GTT TGA TCC TGG CTC AG-3‘) and 1492R (5‘-ACG GCT ACC TTG TTA CGC TT- 3‘) by standard procedure.32 (link) The purified PCR product (~1,426 bp) was sequenced, and the edited sequence data was assembled into a final consensus sequence. The obtained consensus was blasted in the nucleotide National Center for Biotechnology Information (NCBI) GenBank database and submitted into GenBank under accession number (MF671984). The PCR product of aiiA gene (~720 bp) of the same isolate was purified using QIA quick PCR purification kit (Qiagen) and was sequenced. The open reading frame of the lactonase enzyme was obtained using CLC Main Workbench 5 (CLC Bio, Aarhus, Denmark). Alignment of the amino acid sequence of the identified lactonase enzyme with AHL homologous lactonases retrieved from the NCBI database was done using CLC Main Workbench 5. The neighbor-joining method was used to construct the phylogenetic tree using the MEGA 5 software. Cell-free lysate was analyzed by SDS-PAGE and was visualized by Coomassie brilliant blue staining (Sigma-Aldrich).
5
Osteogenic Differentiation of hMSCs
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To test the effect of SCF on odonto/osteogenic differentiation of hMSCs, cells of passage 4-6 were seeded in 96-well plates containing the growth medium described above. Three days after the cells reached 70% confluence, an osteogenic differentiation medium was supplied as previously reported. 22 All measurements were performed in triplicate. The growth medium and the standard differentiation medium were used as a negative and positive control, respectively. Alkaline phosphatase (ALP) activity was assessed quantitatively with a modified assay as described earlier 25 and normalized to total cellular protein concentrations using Coomassie Brilliant Blue staining (Sigma-Aldrich). Furthermore, cells were stained with ALP staining solution, including a mixture of 0.1 mg/mL naphthol AS-MX phosphate and 0.6 mg/mL Fast Blue BB salt (Sigma-Aldrich) and 0.5% N,N-dimethylformamide, 2 mM MgC1 2 in 0.1 M Tris-HCl at pH 8.5.
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