Cells grown in 10-cm Petri dishes were washed twice with ice-cold Dulbecco's PBS and lysed for 30 min on ice with frequent pipetting. Lysis buffer used was 10 mM Tris/Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.5% Nonidet P40 substitute supplemented with 1x protease inhibitor (Roche; cOmplete mini, EDTA free, catalog 4693159001) and 1x phosphatase inhibitor (Thermo Fisher Scientific; PhosStop, catalog 4906845001) cocktails. Lysates were cleared by centrifugation at 17,000 g for 10 min at 4°C. GFP-trap magnetic agarose (Chromotek; catalog gtma-10) was used for eYFP-CEP90 coimmunoprecipitation assays and HA-magnetic beads (Pierce; catalog 88837) for CEP83-HA coimmunoprecipitation assays. Coimmunoprecipitation assays were performed according to manufacturer guidelines. Samples were separated on 4–15% Criterion TGX Precast gels (Bio-Rad) and transferred to Immobilon polyvinylidene fluoride membranes (0.45 µm pore size) for chemiluminescence detection.
Immobilon polyvinylidene fluoride membranes
Manufactured by Bio-Rad
Immobilon polyvinylidene fluoride (PVDF) membranes are a type of microporous membrane used in various laboratory applications. These membranes are designed for the transfer and immobilization of proteins, nucleic acids, and other biomolecules during techniques such as Western blotting, dot blotting, and electroblotting.
Automatically generated - may contain errors
2 protocols using immobilon polyvinylidene fluoride membranes
1
Immunoprecipitation and Western Blot Analysis
2
Coimmunoprecipitation Assay for Protein Interactions
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Cells grown in 10-cm Petri dishes were washed twice with icecold Dulbecco's PBS and lysed for 30 min on ice with frequent pipetting. Lysis buffer used was 10 mM Tris/Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.5% Nonidet P40 substitute supplemented with 1x protease inhibitor (Roche; cOmplete mini, EDTA free, catalog 4693159001) and 1x phosphatase inhibitor (Thermo Fisher Scientific; PhosStop, catalog 4906845001) cocktails. Lysates were cleared by centrifugation at 17,000 g for 10 min at 4°C. GFP-trap magnetic agarose (Chromotek; catalog gtma-10) was used for eYFP-CEP90 coimmunoprecipitation assays and HA-magnetic beads (Pierce; catalog 88837) for CEP83-HA coimmunoprecipitation assays. Coimmunoprecipitation assays were performed according to manufacturer guidelines. Samples were separated on 4-15% Criterion TGX Precast gels (Bio-Rad) and transferred to Immobilon polyvinylidene fluoride membranes (0.45 µm pore size) for chemiluminescence detection.
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