The E. coli cells were collected and washed twice with phosphate buffered saline (PBS, 0.01 M, pH 7.2–7.4) and disrupted using a high-pressure cell disruptor (Constant Systems LTD, UK). The his6-tagged recombinant protein was purified using Ni-NTA His·Bind Column (Novagen). The protein was quantified using a Bradford protein assay kit (Beyotime, China), fractionated on a 12% SDS-PAGE gel and visualized by Coomassie blue staining. For western blot, proteins were transferred to PVDF membrane (Millipore) and blocked using quick block western reagent (Beyotime, China). HRP Anti-6X His tag antibody [GT359] (Abcam, catalog No. ab184607, 1:10,000) was used to detect His6-tagged proteins, and acetyl lysine mouse monoclonal antibody (EasyBio, China, catalog No. BE3411, 1:2000) and Goat anti-mouse IgG (H&L) HRP-conjugated antibody (EasyBio, China, catalog No. BE0102, 1:10,000) was used for protein acetylation analysis. Then protein signal was detected using Immobilon Western HRP substrate (Millipore) and Fusion FX6 Imaging System (Vilber, France).
High pressure cell disruptor
Manufactured by Constant Systems
Sourced in United Kingdom
The High-pressure cell disruptor is a laboratory equipment designed to break open cells and release their contents. It uses high pressure to physically disrupt the cell membranes and walls, exposing the cellular components for further analysis or extraction.
Automatically generated - may contain errors
Lab products found in correlation
Quick block western reagent, by Beyotime (1 mentions)
Fusion fx6 imaging system, by Vilber (1 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Pproex hta vector, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Histrap hp 5 ml, by GE Healthcare (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
6 protocols using high pressure cell disruptor
1
His6-tagged Protein Purification and Analysis
2
Purification of MBP Fusion Proteins
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The genes encoding MBP or MBP fusions were cloned into p416-GALl1 (Mumberg et al., 1994 (link)) and expressed in yeast. About 16,000 OD600 of cells in 100 ml lysis buffer (500 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM DTT, and protease inhibitors) were lysed with a high-pressure cell disruptor (Constant Systems Limited). The lysate was cleared by centrifugation at 10,000 g for 10 min 4°C, filtered, and passed through a column containing amylose (New England Biolabs, Inc.). The column was washed with 500 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM DTT, and 0.1% Mega-8 and eluted with 10 mM maltose, 500 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM DTT, and 0.1% Mega-8.
3
Recombinant PfPLSCR Protein Expression
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Codon-optimised full-length Pfplscr was synthesised as a gBlock® (Integrated DNA Technologies) and cloned into the BamHI/XhoI site of the pPROEX HTa vector (Invitrogen), allowing the expression of recombinant PfPLSCR with an N-terminal hexa-histidine affinity tag (His::PfPLSCR) in E. coli BL21 (DE3) cells. Protein expression was induced with 1 mM IPTG for 14−16 h at 16 °C. Bacterial cells were pelleted and lysed in lysis buffer [250 mM NaCl, 0.5 mM CaCl2, 1 mM MgCl2, 50 mM HEPES pH 7.5] using a high-pressure cell disruptor (Constant Systems). Insoluble material was then centrifuged at 30,000 g (30 min, 4 °C) and washed twice with wash buffer [0.5 M NaCl, 0.5 mM CaCl2, 1 mM MgCl2, 50 mM HEPES pH 7.5] by sonication and centrifugation as described above. Inclusion bodies were solubilised in solubilisation buffer [250 mM NaCl, 0.5 mM CaCl2, 1 mM TCEP, 1% SDS, 25 mM HEPES pH 7.5] under gentle agitation over night at 25 °C and the soluble fraction collected by centrifugation at 30,000 g (30 min, 25 °C). All buffers contained cOmplete™ EDTA-free protease inhibitors (Roche).
4
Purification of Recombinant HMGN1 Protein
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A DNA construct comprising a hexa-histidine-tag followed by a tobacco etch virus (TEV) cleavage site (ENLYFQ|S) and full-length HMGN1 (His6-TEV-HMGN1, Eurofins Genomics, full sequence in Supplementary Data S1) was cloned into a pET21a plasmid via NdeI and XhoI restriction sites. Expression was carried out in E. coli BL21(DE3) using 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) containing 100 µg/mL ampicillin. Overnight cultures were diluted to OD600 ~ 0.2, grown at 37 °C until OD600 ~ 0.7 and protein overexpression was induced with 1 mM isopropyl thiogalactopyranoside (IPTG). After 4 h, cells were harvested and cell pellets resuspended in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) buffer and lysed by passing twice through a high-pressure cell disruptor (Constant Systems). The lysate was centrifuged and the supernatant loaded on a NiNTA column (GE HisTrap HP 5 mL) equilibrated with TBS. Protein was eluted from the column with a gradient of 0-400 mM imidazole in TBS over 60 min. Fractions containing His6-TEV-HMGN1 were identified by SDS-PAGE, pooled and dialyzed against TBS. The His6-tag was removed by TEV protease cleavage at 4 °C overnight with a 1:20 v/v ratio of TEV protease and 1 mM dithiothreitol (DTT). Finally, full length HMGN1 was purified by RP-HPLC on a C4 column with a gradient of 5-40% ACN in water with 0.1% TFA over 35 min at a flow rate of 3 mL/min.
5
Profiling Outer Membrane Proteins
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The analysis of outer membrane protein (OMP) profiles by SDS-PAGE was performed as described by Yethon et al. (2000) with some modifications. After treatment with LPC-058 as described above, equal numbers of cells (standardized by measuring the optical density at 600 nm) of LPC-058–treated and untreated cultures were harvested by centrifugation. After resuspension in 20 mM Tris buffer (pH 8), the cells were lysed by a passage through a High Pressure Cell Disruptor (Constant Systems) at 21,000 psi and centrifuged (3,500 × g, for 10 min) to remove the cell debris. The total membrane fractions were collected by centrifugation (100,000 × g, for 1 h) and then resuspended in 20 mM Tris (pH 8) 2% Sarkosyl buffer. The Sarkosyl-insoluble OM fraction was collected by centrifugation (100,000 × g, for 1 h), then washed with 20 mM Tris buffer (pH 8), and recentrifuged. The resulting pellet was resuspended in 250 μL of 20 mM Tris buffer (pH 8) and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Input samples from each cell culture were processed to obtain the cell crude extract and analyzed by western blot, as described in SDS-PAGE and Immunoblotting.
6
Recombinant C9orf72 Protein Purification
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Cell lysis and protein extraction was performed on ice. All the buffers used for purification were always made fresh and autoclaved prior to use. Cells from ∼4 l of Sf9 cultures weighing (approximately) 14 g was used per purification. Cells were dispersed in 42 ml phosphate-buffered saline (PBS) (30 ml buffer per 10 g of cells) supplemented with 5% glycerol. A high-pressure cell disruptor from Constant Systems Ltd was used to lyse the cells (30 K psi). The lysate was centrifuged at 30,000×g for 45 min at 4 °C. Following centrifugation, 10 μl of Benzonase was added to the clarified lysate and stirred for 20 min at 4 °C. The lysate was then passed through a 0.45 μm filter before being applied to a five ml HisTrap HP column (pre-equilibrated with lysis buffer) at a flow rate of 0.8 ml/min. The column was then washed with 10 column volumes of PBS. Recombinant C9orf72, captured on the column, was eluted using PBS supplemented with 300 mM imidazole (pH 7.4). The eluted protein was concentrated and further purified by size-exclusion chromatography using Superdex200 (GE Healthcare, Buckinghamshire, UK). The eluate absorbance was monitored at 280 nm and fractions corresponding to the major absorbance peak were pooled and concentrated.
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