NLS1–HK2 cDNA was cloned in-frame with BirA* fused to NLS1 into a tetracycline-inducible pcDNA5 FLP recombinase target/tetracycline operator (FRT/TO) expression vector, which was then transfected into Flp-In T-REx HEK293 cells. The cells were collected and pelleted (800g for 3 min), the pellet was washed twice with PBS and the dried pellets were snap frozen. The pellets were lysed in 10 ml of modified RIPA lysis buffer at 4 °C for 1 h and then sonicated (30 s at 35% power; Sonic Dismembrator 500, Fisher Scientific) to disrupt the visible aggregates. The lysate was centrifuged at 35,000g for 30 min. The clarified supernatants were incubated with 30 l packed, pre-equilibrated streptavidin-Sepharose beads (GE) at 4 °C for 3 h on an end-over-end rotator. The beads were collected (800 g for 2 min) and washed six times with 50 mM ammonium bicarbonate (pH 8.3). The beads were then treated with l -1-tosylamide-2-phenylethyl chloromethyl ketone–trypsin (Promega) for 16 h at 37 °C on an end-over-end rotator. After 16 h, another 1 µl of l -1-tosylamide-2-phenylethyl chloromethyl ketone–trypsin was added and the sample was incubated in a water bath at 37 °C for 2 h. The supernatants were lyophilized and stored at 4 °C for downstream mass spectrometry analysis. Two biological and two technical replicates were completed and the NLS1-HK2 interactors were normalized to the BirA* spectral counts.
Sonic dismembrator 500
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sonic Dismembrator 500 is a laboratory instrument designed for the disruption and homogenization of biological samples. It utilizes high-frequency sound waves to physically break down and homogenize a variety of sample types, including cells, tissues, and other solid materials.
Automatically generated - may contain errors
Lab products found in correlation
High performance streptavidin packed beads, by GE Healthcare (2 mentions)
Micro bio spin chromatography column, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Amplex red sphingomyelinase assay kit, by Thermo Fisher Scientific (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Anti h3k27me3 antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg antibody, by Santa Cruz Biotechnology (1 mentions)
Omni th tissue homogenizer, by Omni International (1 mentions)
Allprep kit, by Qiagen (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Gapdh, by Abcam (1 mentions)
Tpck trypsin, by Promega (1 mentions)
Treated trypsin, by Promega (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
20 protocols using sonic dismembrator 500
1
Identification of NLS1-HK2 Interactors
2
Chloroform-Dispersed Single-Walled CNT Ink
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Chloroform s-CNT inks are prepared from isolating s-CNTs from CNT soot using a previously established procedure.43 (link) Briefly, a 1 : 1 ratio by weight of arc-discharge CNT soot (698695, Sigma-Aldrich) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6′-{2,2′-bipyridine})] (PFO-BPy) (American Dye Source, Inc., Quebec, Canada; #ADS153-UV) are each dispersed at a concentration of 2 mg mL−1 in ACS grade toluene. This solution is sonicated with a horn tip sonicator (Fisher Scientific, Waltham, MA; Sonic Dismembrator 500) and then centrifuged in a swing bucket rotor to remove undispersed material. After centrifugation, the supernatant containing polymer-wrapped s-CNTs is collected and centrifuged for an additional 18–24 h to sediment and pellet the s-CNTs. The collected s-CNT pellet is redispersed in toluene with horn tip sonication and again centrifuged. The centrifugation and sonicating process is repeated a total of three times. The final solution is prepared by horn tip sonication of the s-CNT pellet in chloroform (stabilized with ethanol). s-CNTs prepared via this approach are characterized by a log-normal length distribution with an average length of 580 nm and diameters varying from 1.3 to 1.8 nm.44 (link) Concentration of s-CNT ink was determined using optical cross sections from the CNT S22 transition. This solution is referred to as s-CNT ink in the manuscript.
3
Preparation of TCDA Nanoparticles
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10,12-tricosadiynoic acid (1 mM, TCDA, GFS Chemical, 97%) was dissolved in chloroform (2 mL, anhydrous, Sigma-Aldrich, 99%). Then, the solution was filtered through a 200 nm pore syringe filter (Macherey–Nagel). The solution was then dried under N2 gas. Next, deionized water (20 mL) was added to the dried sample, which was heated to 80 °C and sonicated at 10 mW (Sonic Dismembrator 500, Fisher Scientific) for 15 min. This solution was then filtered using an 800 nm pore syringe filter (Merck Millipore) and stored at 4 °C for 6 h.
4
Fabrication of Liposome Particles
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The fabrication of artificial liposome particles was referred to by previous work [36 (link)]. [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, >99%, Avanti Polar Lipids, Alabaster, AL, USA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, >99%, Avanti Polar Lipids), and cholesterol (≥99%, Sigma-Aldrich) were dissolved in chloroform (2 mL). The solution was filtered using a syringe filter (0.2 µm pore, PTFE membrane, Macherey-Nagel, Dueren, Germany) and dried using N2 gas. Deionized water (10 mL) was added to the dried sample and heated in an 80 °C bath for 15 min. Then, the solution was sonicated at 10 mW (Sonic Dismembrator 500, Fisher Scientific, Hampton, USA) for 15 min and stored at 4 °C before use. The unstructured lipids and cholesterols were washed by centrifugation at 12,000 rpm for 10 min.
5
Quantifying Bacterial Sphingomyelinase Activity
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Top10F’ E. coli containing the WT or mutated pTrcHispld plasmids were grown in LB broth overnight at 37 °C. Aliquots of bacteria were centrifuged for 10 min at 9600× g. Bacterial pellets were suspended in lysis buffer and sonicated for 30 s at 40% amplitude a total of three times using a Sonic Dismembrator 500 (Fisher Scientific, Hampton, NH, USA). Lysates were then incubated with a master mix from an Amplex Red Sphingomyelinase Assay kit (Invitrogen, Eugene, OR, USA) (200 µM Amplex Red Reagent, 1 U horseradish peroxidase/mL, 4 U alkaline phosphatase/mL, 0.1 U choline oxidase/mL, and 0.25 mM sphingomyelin in 0.1 M Tris-HCl-10 mM MgCl2). The assay was allowed to incubate at 37 °C for 30 min protected from light. Fluorescent emissions were measured using a FLUOstar Omega plate reader with excitation at 544 nm and detection at 590 nm.
6
Protein Fractionation and Quantification
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All sample transfers and manipulations were carried out in an anoxic chamber and buffers were degassed with N2. Stored frozen cells were allowed to warm to room temperature and 2 μg ml−1 of DNase I were added. The cell suspension was mixed for 30 min. The sample vial was then placed in an ice-water slurry and sonicated for 30 min (Fisher Scientific, Sonic Dismembrator 500). Phase-contrast microscopy confirmed cell lysis. The cell suspension was spun in a centrifuge at 100,000 × g for 45 min. The supernatant was removed as the soluble protein fraction and the pellet was resuspended in buffer after being homogenized with a glass tissue grinder. The suspension was spun at 100,000 × g for 45 min as before and resuspended three times to wash the pellet. After the final spin, the pellet was resuspended in 1 ml of buffer. This was used as the insoluble protein fraction. The protein concentrations of the soluble protein fractions were determined spectrophotometrically using a protein assay kit (Bio-Rad). The protein concentrations of the insoluble protein fractions were determined using the DC protein assay kit (Bio-Rad). Bovine serum albumin was used as the standard for both procedures. Protein fractions that were not used immediately for enzyme activity assays were flash frozen in liquid N2 and stored at −80°C.
7
Chromatin Immunoprecipitation of H3K27me3
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Chromatin immunoprecipitation was performed on the treated uterine stromal cell line and the decidua tissue using the Zymo-Spin ChIP Kit (Irvine, CA). Briefly, the tissue was mechanically homogenized using an Omni TH tissue homogenizer (Omni International, Kennesaw, GA) then all samples were crosslinked using 1% formaldehyde. The crosslinking reaction was stopped using 0.125 M glycine. The samples were then washed with phosphate buffered saline supplemented with protease inhibitors, centrifuged, and stored at −80 °C until ready for use. Next, the samples were sonicated to a size of approximately 250 bp size using a Sonic Dismembrator 500 (Fisher Scientific, Waltham, MA). The size of the chromatin was confirmed by running the sheared chromatin on a DNA 1% Agarose gel. After sonication, the immunoprecipitation reaction was performed using an anti-H3K27me3 antibody (Cell Signaling Technology, Danvers, MA) and a rabbit anti-IgG antibody (Santa Cruz Biotechnology, Dallas, TX) as a negative control and the ZymoMag Protein A beads supplied with the kit. The samples were washed with the provided wash buffers, then reverse crosslinked, and the resulting DNA was purified and stored until they were ready for PCR.
8
Protein Extraction and Digestion from T-cells
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The isolated cells from patients were resuspended in 100 μl of 8 M Urea in 40 mM Tris-HCl (pH 7.6) and pooled with a similar number of total cells between biological replicates. The suspension was sonicated using focus sonicator (Sonic Dismembrator 500, Fisher Scientific) for 3 cycles of 10 s pulse with 10 s intervals at 10% of power. After sonication, a magnetic rack was used to remove the T-Activator beads used for the polarization. Protein concentration was measured using BCA assay and 40 μg of each sample was digested with trypsin using In-solution digestion.
9
Adrenal Protein Isolation and Western Blot
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The All-Prep kit from Qiagen was employed to isolate protein from half of the remaining adrenal gland. Again, homogenization was performed with the TissueLyser (Qiagen). The protein-solubilizing buffer was supplemented with DTT to a total concentration of 8 mg DTT/1 mL ALO buffer to optimize pellet solubilization. ALO (300 μL) was then added to protein pellets, and samples were sonicated for 10 s at 100% amplitude (Sonic Dismembrator 500, Fisher Scientific). Following resuspension, samples were stored at -80°C until western blot analysis could be performed. A total of 5 μL of each sample was used for western blot, and gels were run as performed previously [2 (link)]. Gels were transferred to nitrocellulose membranes. Blots probing for DBH were blocked with 2% BSA. Primary antibodies used include TH (Novus Biologicals), DBH (Abcam), PNMT (Abcam), SP1 (Santa-Cruz), GR (Abcam), and GAPDH (Abcam). Based on primary antibody origin, secondaries conjugated to HRP-IgG were used. Blots were incubated for 2 minutes with ECL and exposed to a film for band visualization as described by Haan and Behrmann [18 (link)]. Quantification was performed using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA), and gels were normalized to GAPDH control. PAH and EGR1 were not quantified as antibodies suitable for western blot were not readily available.
10
Protease Interactor Identification via BioID-MS
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BioID-MS was conducted as described previously (42) . NLN or ClpP complementary DNA (cDNA) was fused in-frame with a mutant Escherichia coli biotin-conjugating enzyme, BirA R118G (or BirA*) into a tetracycline-inducible pcDNA5 FLP recombinase target/ tetracycline operator (FRT/TO) expression vector, which was then transfected into Flp-In T-REx HEK293 Flp-In cells. Cells were lysed, sonicated twice for 10 s at 35% amplitude (Sonic Dismembrator 500; Fisher Scientific), and centrifuged at 35,000g for 30 min at 4°C. Supernatants were passed through a Micro Bio-Spin chromatography column (Bio-Rad 732-6204) and incubated with 30 l of high-performance streptavidin packed beads (GE Healthcare) for 3 hours at 4°C on an end-over-end rotator. Beads were collected (2000 rpm, 2 min) and washed six times with 50 mM ammonium bicarbonate (pH 8.3). Beads were then treated with l-1-tosylamide- 2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Promega) for 16 hours at 37°C on an end-over-end rotator. After 16 hours, another 1 l of TPCK-trypsin was added for 2 hours and incubated in a water bath at 37°C. Supernatants were lyophilized and stored at 4°C for downstream MS analysis. Two biological and two technical replicates were completed for each protease. NLN's interactors were compared to ClpP's interactors, which were normalized to each protease's respective BirA* spectral counts.
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