Microsomal material was prepared and CHAPS washed from α ade−.lys−.leu−.Δaur1−.pESC-LEU_ LmjFIPCS as previously described19 (link),22 (link). Briefly, each compound at the desired concentration was incubated, in at least triplicate, in 96-well plates (Corning) in phosphate buffer (71.4 mM, pH 7.0) with PI (100 µM, final concentration, Avanti Polar Lipids), CHAPS (600 µM, Sigma-Aldrich) and CHAPS-washed microsomal membranes (0.6 U enzyme per reaction) for 15 minutes at 30 °C. NBD-C6-ceramide (5 µM; ThermoFisher) was added, final volume of 40 µl per well, and the plates incubated at 30 °C for a further 25 minutes. Following quenching with 200 µl methanol per well, the reaction product was separated using exchange chromatography in 96-well filter plates (Millipore)19 (link),22 (link) and the fluorescence measured at Ex460/Em540 using a FLx800 Fluorescence Microplate Reader and Gen5 1.08 Data Analysis Software (BioTek). Analyses were carried out using GraphPad Prism 7.
Chaps
Manufactured by Merck Group
Sourced in United States, Germany, Macao, Australia, Sweden, United Kingdom, France, Italy
CHAPS is a zwitterionic detergent used in biochemical applications. It is commonly employed for protein solubilization and purification. CHAPS exhibits a critical micelle concentration of approximately 8-10 mM in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Urea, by Merck Group (40 mentions)
Dithiothreitol (dtt), by Merck Group (38 mentions)
Thiourea, by Merck Group (35 mentions)
Protease inhibitor cocktail, by Merck Group (13 mentions)
Triton x 100, by Merck Group (11 mentions)
Iodoacetamide, by Merck Group (11 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (10 mentions)
Hepes, by Merck Group (10 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (10 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Tween 20, by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (8 mentions)
Glycerol, by Merck Group (7 mentions)
Acrylamide, by Merck Group (7 mentions)
Sodium chloride (nacl), by Merck Group (7 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (7 mentions)
Bromophenol blue, by Merck Group (6 mentions)
Protease inhibitor, by Roche (5 mentions)
Bradford assay, by Bio-Rad (4 mentions)
191 protocols using chaps
1
In Vitro Ceramide Synthesis Assay
2
Transiently Transfected 293TT Cell Lysis
3
Co-Immunoprecipitation Assay for SIRT3 Interactors
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Co-IP assay was carried out as previously reported (37 (link)). In brief, HEK293T cells transfected with plasmids expressing SIRT3-FLAG or Luc-FLAG were lysed in CHAPS lysis buffer (120 mM NaCl, 0.3% CHAPS (Sigma, C900480), 1 mM EDTA, 40 mM HEPES, pH 7.5, and cOmpleteTM protease inhibitor cocktail (Roche)) at 4°C for 2 h with rotation, and then centrifuged at 12 000 g at 4°C for 30 min to collect the supernatants. The supernatants were mixed with anti-FLAG Affinity Gel (Sigma, A2220) and rotated at 4°C overnight. The resulting immunocomplexes were washed with CHAPS lysis buffer three times, incubated with FLAG peptide in CHAPS lysis buffer at 4°C for 2 h with rotation to competitively elute SIRT3-interacting protein complexes by anti-FLAG Affinity Gel, and then centrifuged at 3 000 g at 4°C for 5 min. Finally, the resultant supernatants were boiled in 1× SDS loading buffer for 10 min and processed for western blot analysis.
4
Saliva Collection from Culicoides Midges
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Saliva was collected from the Arthropod-Borne Animal Diseases Research Unit (ABADRU)-maintained C. sonorensis colony (Jones, 1960 ), over a four month period using an artificial membrane feeder system as previously described (Langner et al., 2007 (link)). Briefly, midges were maintained during collection on a 10% sucrose diet. Saliva was collected onto Millipore 0.22 µm hydrophilic membrane discs (Millipore, Billerica, MA) from 3 to 17 day old adult female midges daily. The discs were placed into phosphate buffered saline (PBS) pH 7.4 supplemented with 1 mM 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS; Sigma Aldrich, St. Louis, MO) agitated via vortex for 5 min and stored at 4 °C for up to one week. CHAPS-PBS-saliva solution collected for the period was concentrated using an Amicon Ultra-15 Centrifugal Filter with Ultracel-3 membrane according to manufactures guidelines (Millipore) and then stored at −80 °C until use. Protein concentrations were estimated using a nanophotometer (Implen, Westlake Village, CA).
5
Decellularization of Iliac Artery Graft
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Decellularization of native iliac artery was accomplished as described previously [16 (link)]. Briefly, iliac artery was incubated in 22 h in 3-[(3-Chloamidopropyl)dimethyl- ammonium]-1- propanesulfonate (CHAPS) buffer (8 mM CHAPS [Sigma–Aldrich, St Louis, MO], 1 M NaCl [Sigma–Aldrich], and 25 mM Ethylenediaminetetraacetic acid (EDTA) [Sigma–Aldrich] in PBS), followed by 22 h in Sodium dodecyl sulfate (SDS) buffer containing 1.8 mM SDS (Sigma–Aldrich), 1 M NaCl and 25 mM EDTA in PBS, 48 h of phosphate buffered solution (PBS) washes. We further subjected the decellularized vascular grafts to DNase I (3000 U/ml) and RNase A (3000 U/ml) treatment for 1 h. Decellularized iliac artery was stored in PBS containing penicillin (100 U/ml) and streptomycin (0.1 mg/ml) and placed at 4°C until use.
6
Western Blot Protein Analysis
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Cells were lysed at 4 °C in CHAPS lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm EDTA, 1% (w/v) CHAPS (Sigma-Aldrich), and protease and phosphatase inhibitor mixtures (Roche Applied Science)). The lysates were cleared by centrifugation (15,000 × g for 15 min), resolved on a 12% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The antibody dilutions used were rabbit anti-FLAG (1:1,000; Sigma, F7425), rabbit anti-HA (1:10,000; Sigma, H6908), mouse anti-tubulin (1:10,000; Millipore, 05-829), rabbit anti-YFP/GFP (1:25,000; Abcam, ab290), and anti-SERCA (1:1,000; Calbiochem, 564702). Purified proteins were resolved on Novex 12% Tris/glycine gels (Invitrogen) in the absence of reducing agent and stained with Imperial stain (Pierce) or Coomassie Blue (R-250).
7
Apoptosis Analysis via PARP Cleavage
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Cells were treated with the indicated ABT-199 doses for 24 h. Cells were lysed with CHAPS buffer (10 mmol/L HEPES, 150 mmol/L NaCl, 2 % CHAPS [Sigma Aldrich, MO]) as previously described [19 (link)]. Protein from cell lysate (25 μg per well) was loaded and an anti-PARP antibody (Cell Signaling, MA; #9542) was used to detect PARP cleavage.
8
Decellularized Vascular Graft Development
9
Protein Extraction and 2D-Gel Separation
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Tissue homogenization and 1-DE protein separation were done as previously described (11) .
Briefly, 100 mg of each tissue was homogenized in a detergent solution (4% Triton X100, 1X antiprotease cocktail; Sigma, St Louis, MO, USA) and ground using a grinding kit (GE Healthcare) before protein precipitation with a 2D cleanup kit (GE Healthcare). The supernatant was removed and the pellet was resuspended in 250 ml of sample buffer (8 M urea/2 M thiourea [Sigma], 4% CHAPS [Sigma]). Protein concentration was determined using the Bradford assay (BioRad). Proteins (500 mg per gel) were eluted into rehydration buffer (8 M urea/2 M thiourea [Sigma], 2% CHAPS [Sigma],
DeStreak reagent [15 mg/ml, GE Healthcare] and ampholytes [1% IPG buffer, GE Healthcare]) before first separation according to their isoelectric points along a nonlinear immobilized pH-gradient (IPG) strip (pH 3-11 NL, 18 cm long) using an IPGphor III apparatus (GE Healthcare), as described elsewhere (14) . For the second dimension, equilibrated strips were loaded onto 8-18% SDSpolyacrylamide gels and electrophoresis was performed as in (19) . One preparative gel was stained with CBB G-250 (Sigma) and used for spot cutting and protein sequencing. The remaining gels were electroblotted onto ECL membranes (GE Healthcare).
Briefly, 100 mg of each tissue was homogenized in a detergent solution (4% Triton X100, 1X antiprotease cocktail; Sigma, St Louis, MO, USA) and ground using a grinding kit (GE Healthcare) before protein precipitation with a 2D cleanup kit (GE Healthcare). The supernatant was removed and the pellet was resuspended in 250 ml of sample buffer (8 M urea/2 M thiourea [Sigma], 4% CHAPS [Sigma]). Protein concentration was determined using the Bradford assay (BioRad). Proteins (500 mg per gel) were eluted into rehydration buffer (8 M urea/2 M thiourea [Sigma], 2% CHAPS [Sigma],
DeStreak reagent [15 mg/ml, GE Healthcare] and ampholytes [1% IPG buffer, GE Healthcare]) before first separation according to their isoelectric points along a nonlinear immobilized pH-gradient (IPG) strip (pH 3-11 NL, 18 cm long) using an IPGphor III apparatus (GE Healthcare), as described elsewhere (14) . For the second dimension, equilibrated strips were loaded onto 8-18% SDSpolyacrylamide gels and electrophoresis was performed as in (19) . One preparative gel was stained with CBB G-250 (Sigma) and used for spot cutting and protein sequencing. The remaining gels were electroblotted onto ECL membranes (GE Healthcare).
10
Affinity Purification of Tagged Proteins
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Cells (HEK293) were transfected with the indicated constructs for the expression of GFP- and Flag-tagged proteins. Then, 24 h after transfection, cells were lysed in 600 μL CHAPS lysis buffer [10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate hydrate (CHAPS, Merck, #C3023), 50 mM Tris-HCl (pH7.8), 150 mM NaCl, 5 mM NaF, 0.5 mM phenylmethanesulfonyl fluoride (PMSF) (Merck, #P-7626) and 40 μL/mL cOmplete protease inhibitor cocktail (Roche, #13539320)]. Extracts were incubated with agarose-conjugated anti-Flag antibody (M2, Merck, #A2220) at 4 °C overnight. After washing (6 to 8 times with CHAPS lysis buffer), the precipitates were resuspended in 1× SDS-polyacrylamide gel loading buffer. The plasmids used in this study are given in Table S1 .
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