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.
Fluorescent dye conjugated secondary antibodies
Manufactured by LI COR
Sourced in Germany
Fluorescent-dye-conjugated secondary antibodies are laboratory reagents used in immunochemical techniques, such as Western blotting and immunohistochemistry, to detect and visualize target proteins. These antibodies are designed to bind to primary antibodies and emit fluorescent signals, enabling the identification and quantification of the target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Chaps, by AppliChem (1 mentions)
Lf pa0091, by AbFrontier (1 mentions)
Lf pa0030, by AbFrontier (1 mentions)
Pierce phosphatase inhibitor mini tablets, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nu page lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 polyacrylamide bis tris gels, by Thermo Fisher Scientific (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Complete mini, by Roche (1 mentions)
Sc 7212, by Santa Cruz Biotechnology (1 mentions)
A1978, by Merck Group (1 mentions)
Topoisomerase 1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
6 protocols using fluorescent dye conjugated secondary antibodies
1
Redox-sensitive protein thiol analysis
2
Protein Fractionation and Quantification
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For protein isolation, cells were lysed in a buffer containing 20 mM Tris/cl pH 7.5, 150 mM NaCl, 10 mM NaPPi, 20 nM NaF, 1% Triton, 10 nM okadaic acid (OA), 2 mM orthovanadate (OV), protein inhibitor mix (PIM), and 40 μg/ml phenylmethylsulfonylfluorid (PMSF). Separation of nucleus and cytosol was achieved by lysing the cells in hypotonic buffer (10 nM HEPES pH 7.9, 10 nM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet, 10 mM DTT, protein inhibitor mix (PIM), and 40 μg/ml phenylmethylsulfonylfluorid (PMSF)). Cells were centrifuged at 17000 g, and the supernatant was collected as the cytosolic fraction. The pellet was further lysed with a hypertonic buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, protein inhibitor mix (PIM), and 40 μg/ml phenylmethylsulfonylfluorid (PMSF)). After centrifugation at 17000 g, the supernatant contained most soluble nuclear proteins, while membranes, organelles, and DNA were collected in the pellet. Protein content was determined with the Bradford assay [15 (link)]. Samples were boiled in reducing the Laemmli sample buffer and were subjected to SDS-PAGE followed by Western Blotting. 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
Western Blotting of Whole-Cell Lysates
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Whole-cell lysates were prepared for Western blotting by incubating cells in M-PER mammalian protein extraction reagent (Life Technologies) with protease (Complete Mini, EDTA-free; Roche) and phosphatase inhibitors (Pierce Phosphatase Inhibitor Mini tablets; Thermo Scientific) on ice (10 min). Lysates were centrifuged at top speed in a microcentrifuge for 10 min. Prior to loading onto gels, samples were diluted with NuPAGE sample-reducing agent and NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Life Technologies) and boiled (95°C, 5 min). Following protein separation on NuPAGE 4 to 12% polyacrylamide–bis-Tris gels (Life Technologies) and transfer to nitrocellulose, membranes were probed using the indicated primary antibodies. Fluorescent dye-conjugated secondary antibodies (Li-Cor Biosciences) were used for infrared fluorescence-based detection (Odyssey CLX). Protein levels were quantified by measuring the relative fluorescence intensities of bands (normalized against an actin or GAPDH [glyceraldehyde-3-phosphate dehydrogenase] loading control) using Image Studio 2.1 software.
4
Inflammasome Activation Pathways
5
Isolation and Proliferation Analysis of Murine Lung Endothelial Cells
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Lung endothelial cells were isolated from freshly prepared murine lungs as described before [3] (link). In brief, the tissue was minced and dispase digested at 37 °C. After several washing steps, LECs were separated magnetically using CD144 coated Dynabeads (MACS). LECs were used between passages 5–9. To analyze proliferation cells were seeded in a density of 2×10^6 per cm2 trypsinized and subsequently counted by an automated cell counter (casy, Schärfe) after 1 d of culture.
For protein isolation cells were lysed in a buffer containing 20 mM TRIS/cl pH 7.5, 150 nM NaCl, 10 mM NaPPi, 20 nM NaF, 1% Triton, 10 nM Okadaic acid (OA), 2 mM Orthovanadate (OV), protein-inhibitor mix (PIM) and 40 µg/ml phenylmethylsulfonylfluorid (PMSF). Samples were heated to 95 °C in sample buffer and were transferred on SDS-PAGE followed by Western Blotting. Analysis was performed with an infrared-based detection system using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences. Tert antibody was purchased from Santa Cruz (sc-7212). Telomere length as measured was performed a previously described [1] (link) with adaptation to immunohistochemistry. The PNA-FISH probe TelC probe for a leading strand (repeats of TAACCC) was purchased from PNA Bio Inc (F1001). Experiments were performed according to the manufactures instruction.
For protein isolation cells were lysed in a buffer containing 20 mM TRIS/cl pH 7.5, 150 nM NaCl, 10 mM NaPPi, 20 nM NaF, 1% Triton, 10 nM Okadaic acid (OA), 2 mM Orthovanadate (OV), protein-inhibitor mix (PIM) and 40 µg/ml phenylmethylsulfonylfluorid (PMSF). Samples were heated to 95 °C in sample buffer and were transferred on SDS-PAGE followed by Western Blotting. Analysis was performed with an infrared-based detection system using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences. Tert antibody was purchased from Santa Cruz (sc-7212). Telomere length as measured was performed a previously described [1] (link) with adaptation to immunohistochemistry. The PNA-FISH probe TelC probe for a leading strand (repeats of TAACCC) was purchased from PNA Bio Inc (F1001). Experiments were performed according to the manufactures instruction.
6
Fractionation and Immunoblotting of Cellular Compartments
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For separation of nucleus and cytosol, the cells were lysed in buffer A (10 nM HEPES pH 7.9, 10 nM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet, 10 mM DTT, protein-inhibitor mix (PIM), 40 µg/mL phenylmethylsulfonylfluorid (PMSF). Cells were centrifuged to gain the cytosol containing supernatant. The pellet was cooked in sample buffer to gain the nuclear fraction. Bradford assay was used to determine the protein amount in the cytosolic fraction [19 (link)]. Samples were cooked in sample buffer and were transferred on SDS-PAGE followed by Western Blotting. Identical sample volumes from nuclear and cytosolic fractions were loaded. Analysis was performed with an infrared-based detection system using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences, Bad Homburg, Germany.
Primary antibodies used are: D7L7L from Cell signaling, Danvers, MA, USA for Cre-recombinase, A1978 from Sigma-Aldrich, St. Louis, MO, USA for β-actin and sc-5342 from Santa Cruz Biotechnology, Dallas, TX, USA for Topoisomerase I.
Primary antibodies used are: D7L7L from Cell signaling, Danvers, MA, USA for Cre-recombinase, A1978 from Sigma-Aldrich, St. Louis, MO, USA for β-actin and sc-5342 from Santa Cruz Biotechnology, Dallas, TX, USA for Topoisomerase I.
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