For immunoblotting and detection of MR1, cells were lysed in 0.5% IGEPAL CA-630 (Sigma-Aldrich), 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2 with Complete Protease Inhibitor Cocktail (Roche), and nuclei separated by centrifugation at 13,000 × g for 10 min. Lysates were precleared with normal rabbit serum (Sigma-Aldrich) and protein G-Sepharose then with protein G-Sepharose alone. MR1 was immunoprecipitated using anti-MR1-CT and protein G-Sepharose, and then washed three times with NET buffer (0.5% IGEPAL CA-630, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA) and treated with Endoglycosidase Hf (New England Biolabs) according to the manufacturer’s instructions. Protein samples were denatured with reducing SDS-PAGE sample buffer, separated on NuPAGE 4–12% Bis-Tris precast gel (Life Technologies) and immunoblotted onto nitrocellulose membrane using the iBlot system (Life Technologies). Band density was quantified using FIJI analysis software.
Igepal ca 630
Manufactured by New England Biolabs
Sourced in Japan
IGEPAL CA-630 is a non-ionic detergent that can be used for cell lysis, solubilization of membrane proteins, and as a blocking agent in immunoassays. It is a polyoxyethylene octylphenyl ether with a cloud point around 65°C.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit serum, by Merck Group (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
Endoglycosidase hf, by New England Biolabs (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (1 mentions)
Flag m2 agarose beads, by Merck Group (1 mentions)
2 protocols using igepal ca 630
1
Immunoblotting and Detection of MR1
2
Isolation and Analysis of FLAG-Tagged Protein Complexes
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T-REx 293 cells expressing a FLAG-tagged protein inducibly were harvested, washed twice with ice cold PBS, suspended with extraction buffer (67 mM Tris–HCl, pH 7.4, 200 mM NaCl, 0.1% IGEPAL CA-630 (SIGMA), 1 mM Ribonucleoside–Vanadyl Complex (New England BioLabs), 1 mM PMSF) and lysed by sonication using Bioruptor 200 (highest setting, six times for 30 s, 4°C; CosmoBio, Japan). After centrifugation at 20 000 g for 10 min at 4°C, the supernatant was rotate-incubated with 15 μl of FLAG M2 Agarose beads (Sigma-Aldrich) for 2 h at 4°C. The Agarose beads were washed five times with wash buffer (67 mM Tris–HCl, pH 7.4, 200 mM NaCl, 0.1% IGEPAL CA-630) and eluted for recovery of FLAG-tagged protein complexes with 150 μl of Protein-RNA extraction buffer (7 M urea, 350 mM NaCl, 1% SDS, 10 mM Tris–HCl, pH 8.0, 10 mM EDTA and 2% 2-mercaptoethanol) for 5 min at 25°C. Protein or RNA components were extracted as described in above section ‘Pull down of TDP-43-binding RNA for LC-MS analysis’, and subjected to the immunoblot or northern blot analysis as described below.
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