Cells were seeded in 6-well plates (106 cells/well) and primed for 4 h with 1 μg/ml E. coli O111:B4 LPS in complete DMEM containing 10% FBS. After priming, cells were washed 1 × with serum-free DMEM and 1.1 ml of warm serum-free DMEM was added to each well. Where noted, cells were treated with inhibitors for 15–30 min. Inflammasome activation was triggered by application of freshly prepared 3 mM ATP solution in serum-free DMEM for 30 min. After stimulation, supernatants were collected and spun at 14 000 × g for 15 min at 4 °C to remove cellular debris and approximately 1 ml was transferred to fresh 1.5 ml tubes. Ten microliters of StrataClean resin (Agilent, Santa Clara, CA, USA) was added to each supernatant, mixed well and placed on a rotator in a 4 °C refrigerator for 1 h. Concentrated supernatant protein was collected by pelleting the StrataClean resin, removing the supernatant and heating the resin resuspended in 50 μl 1 × Laemmli buffer at 95 °C for 5 min. Cell lysates were prepared by addition of 100 μl hot 1 × Laemmli buffer to each well for 5–10 min, scraping and transferring samples to 1.5 ml tubes and heating at 95 °C for 5 min.
Strataclean resin
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
StrataClean resin is a high-performance ion exchange resin designed for sample cleanup and analyte enrichment in various analytical applications. It is a sulfonic acid-functionalized resin that can effectively remove interfering ions and concentrate target analytes prior to instrumental analysis.
Automatically generated - may contain errors
Lab products found in correlation
A6001, by Apexbio (3 mentions)
α gfp, by Merck Group (2 mentions)
Superose 6 10 300 gl, by GE Healthcare (2 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Spectra br protein ladder, by Thermo Fisher Scientific (2 mentions)
Mmp 1, by R&D Systems (2 mentions)
Complete mini edta free protease inhibitor, by Roche (2 mentions)
Celltrics filter, by Sysmex (2 mentions)
Phosstop, by Roche (2 mentions)
Ab1791, by Abcam (2 mentions)
Murine m csf, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Gradient station, by BioComp Instruments (2 mentions)
M8823, by Apexbio (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
Pxi imager, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Mouse anti ha 11 epitope tag antibody, by BioLegend (2 mentions)
76 protocols using strataclean resin
1
Inflammasome Activation in Primed Cells
2
Protein Concentration and Immunoblot
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Ten microliters of StrataClean resin (Agilent, Santa Clara, CA, USA) was added to 1 ml of supernatant, mixed well and placed on a rotator in a 4°C refrigerator for 1 h. Concentrated supernatant protein was collected by pelleting the StrataClean resin, removing the supernatant and heating the resin resuspended in 50 μl 1 × Laemmli buffer at 95°C for 5 min, followed by short spin and loading of the sample, without resin, for immunoblot analysis.
3
Proteolytic Activity Assay for IGFBP-3 and IGFBP-5
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Media samples from AGSGR cells were concentrated using StrataClean resin (Agilent Technologies, Stockport, UK) and processed as previously described.7 (link) To assess proteolytic activity, proteins were separated on 15% sodium dodecyl sulfate–polyacrylamide gels and probed using either polyclonal goat IGFBP-3 or IGFBP-5 primary antibodies (R&D Systems) at 1:500 followed by rabbit anti-goat horseradish peroxidase–conjugated secondary antibody at 1:5000 (R&D Systems). Membranes were developed using Supersignal (Thermo Fisher) and chemiluminescence was detected using a Bio-Rad ChemiDoc XRS+ (Bio-Rad). Densitometry was performed using ImageLab software (version 3.0; Bio-Rad, Hertfordshire, UK).
4
Western Blotting of Cellular Proteins
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DCs were lysed using lysis buffer with Pierce protease and phosphatase inhibitors. For cell lysate analysis, protein levels were quantified using the Pierce BCA Assay kit (Thermo Fisher, Waltham, MA) and normalized to 20–30 µg of total protein (depending on the individual blot) prior to running on 12% SDS‐PAGE gels and subsequent transfer to nitrocellulose membranes. For cell supernatant analysis, 2–4 million cells were stimulated in 2 mL of media and supernatant were concentrated 10‐fold using StrataClean Resin (Agilent, Santa Clara, CA) to nonspecifically concentrate all proteins in the supernatant. Cleaved caspase‐1 and cleaved IL‐1β blots were performed on these concentrated supernatant preparations.
5
Fractionation of Nuclear Extracts Containing EDS1-YFP and TRB1-GFP
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Nuclear extracts (600 μl) from the pEDS1:EDS1-YFP complementation line were processed as for IP input in the subsection “Immunoprecipitation (IP) of EDS1-YFP and TRB1-GFP from respective Arabidopsis complementation lines”. Obtained samples were fractionated on the column Superose 6 10/300 GL (50 kDa–5 MDa range, GE Healthcare Life Sciences, Ӓkta FPLC) at the rate 0.5 ml/min in 20 mM Tris–HCl pH 7.4 and 150 mM NaCl. The temperature was kept at 4 °C. In total, 28 0.5 ml fractions per sample were collected, concentrated with StrataClean resin (400714, Agilent) and analysed using Western blot method (α-GFP, 11814460001, MilliporeSigma) with the same total EDS1-YFP sample on each blot for the between-blot normalisation. A high-molecular weight marker was run prior to each experiment (28403842, GE Healthcare Life Sciences).
6
Isolation and Fractionation of Cellular Complexes
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HeLa cells were harvested and mechanically lysed as described above. Cell powder was solubilized in ice-cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 10 mM MgCl2, 0.1% [v/v] Igepal CA-630) supplemented with cOmplete EDTA-free Protease Inhibitor Cocktail (Roche). Lysates were vortexed briefly and incubated on a rotating shaker for 10 min at 4 °C. Lysates were then homogenized with 5 up and down strokes through a 27-gauge needle and cleared by centrifugation at 100,000 g for 1 h at 4 °C. 3–4 mg total protein was loaded onto linear gradients of 10–30% [w/v] glycerol dissolved in 20 mM Tris-HCl pH 8.0, 200 mM K(OAc), 5 mM Mg(OAc)2, 1 mM DTT supplemented with cOmplete EDTA-free Protease Inhibitor Cocktail. Samples were centrifuged at 229,900 g for 18 h at 4 °C using a swinging bucket rotor (Hitachi). Fractions were eluted from the top of the gradient using a density gradient fractionation system (Brandel). During elution, fractions of ~900 μl were collected. Fractions were then supplemented with 300 μl 20 mM Tris-HCl pH 7.5 and 15 μl StrataClean Resin (Agilent) and incubated overnight on a rotating shaker at 4 °C. Proteins were eluted from the resin with 2x SDS sample buffer (100 mM HEPES pH 7.4, 4% [w/v] SDS, 20% [v/v] glycerol, 200 mM DTT, bromophenol blue).
7
Characterizing H. pylori-Induced Inflammatory Response
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MKN28 or AGS human gastric epithelial cells were seeded into 24 wells at 5 x 104 cells/well 24 h before experiments for assessment of cytokine, or 25 cm2 flasks at 7 x 105 cells/flask for secretion experiments. Relevant H. pylori strains were added to cell monolayers at a multiplicity of infection (MOI) of 50:1 unless otherwise stated. Sampled cell culture supernatants were assessed for IL-8 or TNF-α levels using Human IL-8 CytoSet or Human TNF-α ELISA kits (Invitrogen). Cell culture supernatants for protein analysis were 0.2 μM filtered prior to 5 min incubation with 5 μl Strataclean resin (Agilent Technologies). Resin was pelleted by centrifugation, resuspended in 2X sample loading buffer then boiled for 5 min in preparation for 12% SDS-PAGE/Western immunoblot analysis using anti-GSK-3β (Cell Signalling Technology Inc), anti-GAPDH or anti-CagA (Santa-Cruz Biotechnology) antibodies.
8
Western Blot Analysis of Strep-Tagged Proteins
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For Western blot analysis, the supernatants were thawed on ice and precipitated by 10 μl Strata clean resin (Agilent, Waldbronn). For SDS PAGE, the precipitated proteins were dissolved in 50 μl sample loading dye (3 X Laemmli beta‐mercaptoethanol), boiled for 7 min and stored on ice. 10 μl of each sample was loaded on 12% SDS‐PAGE. After gel electrophoresis proteins were transferred onto nitrocellulose membrane (Protran, GE, Frankfurt am Main) using Trans Blot device (Bio‐Rad, Munich) at 350 V for 40 min. Membranes were then blocked overnight with RotiBlock blocking reagent (Roth, Karlsruhe). Membranes were incubated with primary antibody (anti‐strep‐tag rabbit IgG, Abcam) for 1 hr and subsequently with secondary antibodies (anti‐rabbit IgG goat IgG alkaline phosphatase conjugated, Sigma) for 1 hr each at room temperature under gentle shaking. Detection was carried out using BCI/NBP (Sigma, Munich); blots were scanned by Epson scanner.
9
Quantifying Fibrotic Lung Protein Levels
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Fibroblasts were cultured as for RNA analysis. After incubation, cells were lysed into SDS sample buffer for SDS-PAGE and cell conditioned media clarified by centrifugation before concentrating secreted proteins using StrataClean resin at 1:10 (Agilent Technologies, Wokingham, UK) [54 (link)]. For murine fibrosis studies, right lungs were snap frozen and then homogenized in normal saline containing protease inhibitors (Roche, Burgess Hill, UK) before solubilization in SDS sample buffer for SDS-PAGE.
Western blotting of cellular or lung lysates was performed for acetyl histone H3, 1:20000 (Merck Millipore, Watford, UK), and of cell-conditioned medium for secreted pro-LOX and LOX, (1:20, Sigma-Aldrich, Poole, UK) using enhanced chemiluminescence detection (ECL+, GE Healthcare, Buckinghamshire, UK). In the case of acetyl H3, membranes were stripped and reprobed using pan histone H3 antibodies (Merck Millipore, Watford, UK); for LOX quantitation, image analysis of Ponceau Red stain (Sigma-Aldrich, Poole, UK) was used a loading control. Densitometry was used for semi-quantitative analysis of the data.
Western blotting of cellular or lung lysates was performed for acetyl histone H3, 1:20000 (Merck Millipore, Watford, UK), and of cell-conditioned medium for secreted pro-LOX and LOX, (1:20, Sigma-Aldrich, Poole, UK) using enhanced chemiluminescence detection (ECL+, GE Healthcare, Buckinghamshire, UK). In the case of acetyl H3, membranes were stripped and reprobed using pan histone H3 antibodies (Merck Millipore, Watford, UK); for LOX quantitation, image analysis of Ponceau Red stain (Sigma-Aldrich, Poole, UK) was used a loading control. Densitometry was used for semi-quantitative analysis of the data.
10
Quantitative Secretome Analysis of ATII Cells
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