The decellularization of the hUAs was carried out according to previously described protocols, [17 (link)]. Briefly, hUAs (n = 44, l = 2 cm), were incubated in CHAPS solution (8 mM CHAPS (APPLICHEM, Darmstadt, Germany), 1 M NaCl, and 25 mM EDTA (Ethylenediaminetetraacetic acid) in PBS 1×; (Sigma-Aldrich, Darmstadt, Germany) at pH 8 for 22 h, followed by brief washes in PBS 1×. The hUA were further incubated in SDS solution (1.8 mM SDS (Sigma-Aldrich, Darmstadt, Germany), 1 M NaCl, and 25 mM EDTA in PBS 1×) at pH 7.5 for 24 h, followed by 3 washes for 5 min in PBS 1×, to completely remove the detergent. Finally, the arteries were incubated at 37 °C for 48 h in alpha- Minimal Essential Medium (α-MEM, Gibco Life Technology, Darmstadt Germany), containing 40% (v/v) fetal bovine serum (FBS, Gibco Life Technology, Darmstadt, Germany) and 1000 U/mL penicillin-streptomycin (Gibco Life Technology, Darmstadt, Germany). All steps were performed under agitation and sterile conditions.
Chaps
Manufactured by AppliChem
Sourced in Germany
CHAPS is a zwitterionic detergent commonly used in biochemistry and molecular biology applications. It is effective in solubilizing and stabilizing proteins, and is often used in the preparation of protein samples for analysis.
Automatically generated - may contain errors
Lab products found in correlation
C18 ziptip, by Merck Group (3 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Sodium thiosulfate, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Serva Electrophoresis (2 mentions)
Glycerin, by Merck Group (2 mentions)
Formic acid, by BASF (2 mentions)
Bromophenol blue, by Carl Roth (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Thiourea, by Merck Group (2 mentions)
Urea, by AppliChem (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
H 2kb specific antibody y3, by GE Healthcare (2 mentions)
Cyanogen bromide activated sepharose 4b, by GE Healthcare (2 mentions)
Centricons with a 10 kda cut off membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
14 protocols using chaps
1
Decellularization of Human Umbilical Arteries
2
Redox-sensitive protein thiol analysis
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For Prx2, Prx3 and Prx4 cells were incubated with NEM (50 mM) and washed with PBS- NEM (100 mM). Cells were scraped in alkylation buffer (40 mM Hepes, 50 mM NaCl, 1 mM EGTA, Inhibitors, Catalase, 100 mM NEM) and 1% CHAPS (from Applichem) for solubilization. For Trx1 and Trx2 cells were scraped in 20% TCA followed by two acetone washing steps. Proteins were dissolved in EB buffer (10% SDS, 150 mM NaCl, 50 mM Hepes) and incubated with 15 mM AMS (from Life Technologies) for 3 h. Protein amount was determined by Lowry protein assay. Samples were substituted with sample buffer (8.5% glycerin, 2% SDS, 6.25% TRIS/HCl pH 6.8, 0.013% bromphenol blue) and separated on a non-reducing SDS-PAGE gel, followed by Western blot analysis and detection by antibodies. Primary antibodies against Prx2 (#LF-PA0091), Prx3 (#LF-PA0030), Prx4 (#LF-PA0009), Trx1 (#LF-PA0187) and Trx2 (#LF-PA0012) were diluted 1:1000 and were purchased from AbFrontier. The antibody against Nox4 was a gift from Ajay Shah from Kings College London and was diluted 1:1000. After incubation with first antibodies, membranes were analyzed with an infrared-based detection system, using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences.
3
Proteomic Analysis of Cellular Proteins
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Reagents and their sources were as follows: phosphate buffer saline (PBS) (PAA Laboratories, Cölbe, Germany), sodium carbonate, ammonium bicarbonate, thiourea, dithiothreitol (DTT), urea, trypsin, trifluoroacetic acid (TFA) (Sigma-Aldrich), CHAPS (AppliChem, Darmstadt, Germany), Acetonitril (ACN) (Promochem, Wesel, Germany), Immobilized pH gradient strips (IPG strips), ampholytes, protein assay kit (Bio-Rad, Munich, Germany), protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany), sodium dodecyl sulfate (SDS) (Serva, Heidelberg, Germany), potassium ferricynaide, Glycerin, sodium thiosulfate (Merck, Darmstadt, Germany), formic acid (BASF, Ludwigshafen, Germany), bromophenol blue (Carl Roth, Karlsruhe, Germany).
4
Isolation and Purification of MHC-Peptide Complexes
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As described [38 (link),65 (link)], frozen cells were thawed into 10 mM CHAPS (AppliChem, St. Louis, Missouri) in PBS (Gibco, Carlsbad, California) with complete protease inhibitor (Roche, Basel, Switzerland), and class II molecules were isolated by standard immunoaffinity purification using purified monoclonal antibody 2G11 [66 (link)] produced in-house and covalently linked to CNBr-activated Sepharose (GE Healthcare, Chalfont St Giles, UK). MHC–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (TFA; Merck, Whitehouse Station, New Jersey). Peptides were purified by ultrafiltration using centrifugal filter units (Amicon; Millipore, Billerica, Massachusetts), desalted using ZipTip C18 pipette tips (Millipore), and eluted in 35 μl 80% acetonitrile (Merck), 0.2% TFA, then vacuum-centrifuged and resuspended in 25 μl 1% acetonitrile, 0.05% TFA, and stored at −20°C.
5
Immunoprecipitation and Mass Spectrometry
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Dithiothreitol, iodoacetamide, paraformaldehyde (PFA), CPT, proteases and phosphatases inhibitors cocktails were from Sigma-Aldrich. CHAPS was from Applichem (Darmstadt, Germany). DDP was from Pfizer (New York, NY, USA). Rabbit polyclonal antibody against SLIRP (Abcam Cambridge, UK), mouse monoclonal antibody against FLAG peptide (Sigma-Aldrich), mouse monoclonal against β-actin (Sigma-Aldrich), mouse monoclonal antibody against bcl-2 (Santa Cruz Biotechnology, Inc, Dallas, TX, USA) and rabbit polyclonal antibody against bcl-2 (Santa Cruz Biotechnology) were used. Lipofectamine2000 and BCA reagents were purchased from Invitrogen. Enhanced chemiluminescence (ECL) reagents from Pierce Biotechnology (Rockford, IL, USA), Protein A and Protein G agarose beads from Amersham Biosciences Europe (Milan, Italy), sequencing-grade modified trypsin from Promega (Madison, WI, USA) were used. Acetonitrile (ACN), formic acid (FA) and water for mass spectrometry were from Sigma. Water used in this study was deionized using a Milli-Q purification system (Millipore, Billerica, MA, USA).
6
Isolation of HLA-I Molecules
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HLA-I molecules from cell pellets were isolated using the standard immunoaffinity purification procedure as previously described [20 ,21 (link)]. Briefly, frozen cells were lysed using 10 mM CHAPS (AppliChem, Darmstadt, Germany) buffer prepared in PBS, containing a complete protease inhibitor (Roche, Mannheim, Germany). The cell lysates were homogenized by applying pulsed sonification. Thereafter, the HLA-I molecules were purified from lysates by immunoaffinity chromatography with the pan-HLA class I specific W6/32 antibody coupled to CNBr-activated sepharose (GE Healthcare, Chicago, IL, USA). HLA-associated peptides were eluted with 0.2% TFA followed by ultrafiltration of the eluate using 3-kDa Amicon filter units (Merck Millipore, Billerica, MA, USA). Desalting and concentration steps were accomplished by ZipTip C18 (Merck Millipore) and 0.1% TFA, and elution was performed with 32% Acetonitrile (AcN)/0.2% TFA. The final volume of the eluate was reduced by vacuum centrifugation.
7
Protein Solubilization and Quantification
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The precipitates of 1 I cell culture supernatant/conditioned media were incubated in 200 μl DIGE lysis buffer (30 mM Tris–HCl buffer, pH 8.5, containing 7 M urea (Applichem, Darmstadt, Germany), 2 M thiourea (Sigma-Aldrich) and 4% (w/v) 3 [(3 cholamidopropyl)-dimethylamino] 1 propane sulfonate (CHAPS; Applichem)) at 25°C for 30 min thereby vortexing thoroughly. Solubilized proteins were then sonicated using two cycles of five impulses (0.5 s/impulse) at 100% power (Bandelin UW 2070 sonicator, MS 73 needle; Bandelin, Berlin, Germany) and total protein concentration of samples was determined as previously described [52 (link)].
8
Isolation of H-2Kb MHC Class I Molecules
9
Isolation of H-2Kb MHC Class I Molecules
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For H-2Kb isolation, sarcoma cell lines d42m1-T3 and F244 were each expanded to 5 x 108 cells. Prior to harvesting, cells were stimulated with 300 U ml−1 IFN-γ for 48 hours to increase MHC expression. Detachment of cells was facilitated by incubation with 100 U ml−1 Collagenase IV (Gibco) in PBS for 10 min at 37° C in a 5% CO2 incubator. Cells were washed twice with PBS and cell pellets were subsequently snap frozen. MHC class I molecules were isolated as previously described40 (link). In brief, cell pellets were taken up in 1 ml of lysis buffer (1.2% CHAPS (Applichem, Darmstadt, Germany), 1x Protease Inhibitor Cocktail (Roche) in PBS) and homogenized by sonication. Lysates were cleared from remaining cell debris by centrifugation (2,500g, 30 min) and passing through a 0.2 μm filter (Sartorius, Goettingen, Germany). MHC class I molecules were isolated by immunoaffinity purification using H-2Kb-specific antibody Y3 covalently coupled to cyanogen bromide-activated sepharose 4B (GE Healthcare). MHC molecules were eluted with 0.2% trifluoroacetic acid (TFA) and released peptides were further isolated by ultrafiltration through centricons with a 10 kDa cut-off membrane (Millipore, Schwalbach, Germany). Prior to LC-MS analysis peptides were desalted using C18 Zip Tips (Millipore) according to manufacturer’s instructions and volumes were adjusted by vacuum centrifugation.
10
Proteome Analysis of 6-MP and 6-TG
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All cell culture reagents, RPMI, fetal calf serum (FCS), phosphate buffered saline (PBS), penicillin, and streptomycin, were purchased from PAA Laboratories, Colbe, Germany. Acetonitrile (ACN) was purchased from Promochem, Wesel, Germany. CHAPS was obtained from Applichem, Darmstadt, Germany. Urea, thioUrea, dithiothreitol (DTT), trypsin, trifluoroacetic acid (TFA), sodium carbonate, ammonium bicarbonate, 6-MP, 6-TG, and DMSO were obtained from Sigma-Aldrich, Steinheim, Germany. Ampholytes, protein assay kits, and immobilized pH gradient strips (IPG strips) were obtained from Bio-Rad, Munich, Germany. Protease and phosphatase inhibitor cocktails were purchased from Roche, Mannheim, Germany. Glycerin, potassium ferricyanide, and sodium thiosulfate were purchased from Merck, Darmstadt, Germany. Formic acid was from BASF, Ludwigshafen, Germany. Bromophenol blue and Trizma base were from Carl Roth, Karlsruhe, Germany. Sodium dodecyl sulfate (SDS) was purchased from Serva, Heidelberg, Germany.
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