Whole-brain or cortex samples for western blotting were prepared by homogenization of brain tissue (whole brain for p0 animals; one cortex for p14–p21 animals and aged animals) as detailed in Supplemental Experimental Procedures . For Trap-GFP immunoprecipitation, HeLa cells were transfected with endophilin (1, 2, or 3)-EGFP and mCherry/FBXO32-mCherry using Lipofectamine 3000 (Invitrogen). We used Chromotek Trap-GFP agarose beads (Chromotek) and followed manufacturer’s protocol. Alternatively, HeLa cells were transfected with FBXO32-EGFP and coupled to Chromotek Trap-GFP agarose beads. Purified endophilin-1 was added, and all fractions (input, non-bound, and bound) were collected. Samples were subjected to SDS-PAGE electrophoresis and immunoblotted against indicated proteins.
Chromotek gfp trap agarose beads
Manufactured by Proteintech
Sourced in Germany
ChromoTek GFP-Trap Agarose beads are a highly specific and efficient tool for the isolation and purification of GFP-fusion proteins from various sample types. The beads are coated with a single-domain antibody fragment that binds to GFP, allowing for the capture and enrichment of GFP-tagged proteins from cell lysates or other solutions.
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3 protocols using chromotek gfp trap agarose beads
1
Endophilin and FBXO32 Interaction Assay
2
Purification and Immunoblotting of AGP2-GFP
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Total cell extracts of yeast cells untreated or treated with CHX were incubated in ChromoTek GFP-Trap Agarose beads (Proteintech) for 2.5 h at 4 °C to pull-down AGP2-GFP tagged-associated proteins. After incubation, the beads coupled to proteins were washed with washing buffer (10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 150 mM NaCl and 0.05% NonidetTM P40 Substitute) 3 times and collected on a magnetic stand. The proteins were eluted in 2X SDS loading dye and heated at 65 °C for 10 min. Proteins were processed on 12% SDS gel and the membranes were probed with an anti-GFP antibody or Anti-Ubiquitin antibody.
3
Antibody Labeling and Immunoprecipitation
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The full list of antibodies used in this study is provided in the Supplementary Table S1 .
Secondary HRP-labelled antibodies were from Pierce (ThermoFisher Scientific, Waltham, MA, USA). Chromotek GFP-Trap agarose beads were purchased from Proteintech (Planegg-Martinsried, Germany).
Secondary HRP-labelled antibodies were from Pierce (ThermoFisher Scientific, Waltham, MA, USA). Chromotek GFP-Trap agarose beads were purchased from Proteintech (Planegg-Martinsried, Germany).
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