μmacs gfp tagged protein isolation kit

The Co-IP LD isolation method was performed using a μMACS GFP-tagged protein isolation kit (Miltenyi Biotec, Gladbach, Germany) following the method of a previous study (Shimada et al., 2014 (link)). Four-week-old hise1-2/pLDAP3:LDAP3-GFP plants (0.5 g fresh weight) and wild-type plants expressing cytosolic GFP alone were ground with 1.5 mL extraction buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl₂, and 1 mM MgCl₂ using a pestle and mortar. The extracts were centrifuged at 1,000 g for 5 min at 4°C followed by 8,000 g for 10 min at 4°C. The supernatants (1 mL each) were subjected to immunoprecipitation with 50 μL of anti-GFP microbeads (μMACS GFP-tagged protein isolation kit). The samples were incubated at 4°C for 30 min and applied to a μ column (Miltenyi Biotec). The column was washed with 1 mL of the extraction buffer. Pure immunoprecipitates were eluted with 50 μL of sample buffer (100 mM Tris-HCl pH 6.8, 4% [w/v] SDS, 12% [v/v] 2-mercaptoethanol, and 20% [v/v] glycerol) and defined as isolated LDs.

Co-IP assays were performed using a μMACS GFP-tagged protein isolation kit (Miltenyi Biotec). Three-week-old A. thaliana plants (0.5 g fresh weight) expressing MYOB2-GFP or MYOB14-GFP were ground in 1 mL of lysis buffer (150 mM NaCl, 1% [v/v] Ecosurf EH-9, and 50 mM Tris-HCl pH 8.0). The extracts were centrifuged at 1,000 × g for 1 min at 4°C. This was followed by centrifugation of the supernatants at 15,000 × g for 10 min at 4°C. A 1-mL aliquot of each supernatant was subjected to Co-IP with 50 μL of anti-GFP microbeads. Samples were incubated at 4°C for 30 min and applied to a μ column (Miltenyi Biotec). The column was washed with 1 mL of lysis buffer and 0.2 mL of 20 mM Tris-HCl, pH 7.5. Pure immunoprecipitates were eluted with 50 μL of sample buffer (100 mM Tris-HCl pH 6.8, 4% [w/v] SDS, 12% [v/v] 2-mercaptoethanol, and 20% [v/v] glycerol).