ID-IEF using slab gel electrophoresis was performed on 10 µg of total myofibrillar protein as previously described with modification [18 ]. Briefly, IEF gel slabs (0.75 mm thick) containing 5% acrylamide/bis (37.5:1, Biorad, Hercules, CA), 9.1 M urea, 2% (w/v) Triton X-100, and 0.05× of Pharmalyte 4.2–4.9 (GE Healthcare, Chicago, IL), with the catalysts ammonium persulfate and N,N,N,N-tetramethylethylenediamine (TEMED) added separately. Myofibrillar protein samples were prepared in IEF solubilization buffer containing 9.2 M urea (Biorad, Hercules, CA), 2% Triton X-100, 0.02× of Pharmalyte 4.2–4.9, Tributyl Phosphine (Biorad, Hercules, CA) and 0.01% Bromophenol Blue (w/v). Electrophoresis was performed using 40 mM lysine as the cathode buffer and 7 mM phosphoric acid as the anode buffer. The gel was transferred in 0.7% acetic acid (pH 3) in preparation for western blotting.
Tributyl phosphine
Manufactured by Bio-Rad
Sourced in United States
Tributyl Phosphine is a chemical compound used as a reducing agent in various laboratory applications. It functions by donating electrons to other chemical species, facilitating reactions and transformations. The core purpose of Tributyl Phosphine is to serve as a versatile reagent in controlled laboratory settings, without further interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Bis acrylamide, by Bio-Rad (1 mentions)
Triton x 100, by GE Healthcare (1 mentions)
Urea, by Bio-Rad (1 mentions)
Non fat dry milk powder, by Merck Group (1 mentions)
Anti rabbit igg na934v, by GE Healthcare (1 mentions)
Amicon ultra 4 centrifugal filter, by Merck Group (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Las 4000, by Cytiva (1 mentions)
2 protocols using tributyl phosphine
1
Myofibrillar Protein Analysis via IEF
2
Proteomic analysis with PLA2 antibodies
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Amicon Ultra-4 centrifugal filters (3kDa cut-off) came from Merck Millipore. Constituents of the Protease inhibitor cocktail [41 (link),57 (link)] were purchased from Sigma-Aldrich (St. Louis) and AMRESCO Inc. (Dallas, TX, USA). All electrophoresis grade chemicals used in two-dimensional gel electrophoresis (2DE) were purchased from AMRESCO Inc. 3-10NL IPG strips, tributyl phosphine and Polyvinylidene difluoride (PVDF) membrane (0.2 µM pore size) were from Bio-Rad (Hercules, CA, USA). Anti-human rabbit polyclonal iPLA2 antibody (PA5-27945) and anti-human mouse monoclonal sPLA2 antibody (ab-24498) were from Invitrogen and Abcam (Cambridge, UK), respectively. Blocking agents―non-fat dry milk powder and BSA―were from Devondale (Saputo Dairy, Australia) and Sigma Aldrich, respectively. HRP linked anti-mouse IgG (NA93IV) and anti-rabbit IgG (NA934VS) antibodies came from GE Healthcare (Buckinghamshire, UK). Lumunata Cresendo Western HRP substrate was purchased from Merck Millipore. The blots and gels were imaged using the Image Quant™ LAS 4000 Biomolecular Imager (GE Healthcare, Chicago, Il, USA).
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