Membrane vesicles were purified from the strains that overexpress either FtsH or SaeQ. To overexpress FtsH, the vector pYJ335 was PCR-amplified with the primers P371/372, while the ftsH gene including the promoter sequence was amplified with the primers P39/370 (Supplementary Table 8 ). The plasmid was assembled with LIC as described above. The plasmid was inserted into E. coli DH5a, and then into S. aureus RN4220. Finally, the plasmid was transduced by ϕ85 into wild type of S. aureus Newman. For SaeQ protein, we used the NMΔftsH strain with pYJ-SaeQ-his plasmid. The membrane vesicles were prepared from the test strains as described previously30 (link). The protein concentration in the membrane vesicle suspension was determined by the bicinchoninic acid assay (Yeasen Bio, China). The membrane vesicles were stored at -80 °C until use.
Bicinchoninic acid assay
Manufactured by Yeasen
Sourced in China
The bicinchoninic acid (BCA) assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It is based on the reduction of copper(II) to copper(I) by protein in an alkaline medium and the subsequent chelation of the copper(I) ion with two molecules of bicinchoninic acid, forming a purple-colored complex that absorbs light at 562 nm.
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4 protocols using bicinchoninic acid assay
1
Purification and Characterization of Membrane Vesicles
2
Immunoblotting Using RIPA Lysis and ECL Detection
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For immunoblotting, the cells were lysed in RIPA buffer containing protease inhibitors (Bimake; No. B14002) and phosphatase inhibitors (Bimake; No. B15003). Protein concentrations were detected by bicinchoninic acid assay (Yeasen Biotechnology; No. 20201ES90). Then, the proteins were resolved by SDS-PAGE gel and transferred to PVDF membranes (EMD Millipore; No. IPVH00010). After blocking with 5% bovine serum albumin (Sigma; No. V900933-1KG), the membranes were incubated at 4 °C overnight with the indicated primary antibodies, including Flag (1:5000, Sigma; No. GNI4110-FG), MUTYH (1:1000, Abcam; No. ab228722) and Vinculin (1:3000, Sigma; No. V9131). After washing with PBS three times the next day, the membranes were incubated with horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (1:5000, Cell Signaling Technology; No. 7076V and 7074V, respectively). Finally, the protein bands were visualized using an enhanced chemiluminescence substrate kit (Yeasen Biotechnology; No. 36208ES80).
3
Cloning and Purification of SarX Protein
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The isolates S0385 (GenBank accession number AM990992) and TW20 (GenBank accession number FN433596) respectively served as the reference genomes of CA-SA ST398 and HA-SA ST239 strains. The sarX gene was conserved between both of the reference genomes. Genomic DNA extracted from CA-SA ST398 isolate S0385 (GenBank accession number AM990992) served as the PCR template. The sarX gene was amplified using the forward primer 5′- CGGGATCCTTGAATACTGAGAAATTAGAAACAT-3′ and the reverse primer 5′-CGGAATTCTTAAATATTTAAAAATTGTTCTACA-3′. The respective PCR product was digested with BamHI and EcoRI. The PCR product was ligated into pET28a. The resulting plasmid were transformed into Escherichia coli Top10 strain. The correct nucleotide sequence was confirmed by sequencing. The resulting construct was transformed into Escherichia coli strain BL21 (DE3) for isopropyl-β-D-1-thiogalactopyranoside (IPTG)-induced expression according to the manufacturer’s instructions. His-tagged SarX protein was respectively affinity-purified from cleared lysates with Ni-NTA resin (Qiagen) according to manufacturer’s construction. The protein concentration was determined by the bicinchoninic acid assay (Yeasen Bio, China).
4
Protein Expression Analysis of FB-Treated Cells
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The cells were treated with FBs for 48 h, then washed once with phosphate buffered saline and harvested. The harvested cells were lysed for 30 min and rotated at 13,000 rpm/min for 20 min at 4 °C. The soluble portion was gathered and the protein concentration was determined by a bicinchoninic acid assay (YEASEN Biotechnology). Then, the 15 μL protein samples (the protein content of each sample was the same) and pre-stained molecular weight markers were subjected to 10% or 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (this gel had 15 lanes) and transferred to NC membranes (Beyotime Biotech) by electroblotting at 4 °C. Subsequently, the membranes were sealed with 10% skimmed milk powder in Tris-buffered saline and Tween 20 for 2 h at room temperature and incubated overnight in specific antibodies (Actin, ATF4, CHOP, Bip and PERK) (Cell Signalling Technology) diluted 1:1000 or 1:2000 at 4 °C. The secondary antibody IgG was labeled with horseradish peroxidase (Cell Signalling Technology), diluted to 1:2000, and incubated with the membrane at room temperature for 2 h. The staining protein was shown by the Tanon-5200Multi electroluminescence detection system (Tanon).
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