Anti gfp antibody

Size exclusion chromatography was performed as previously described [89 (link)]. Elution fractions were either analyzed by immunodetection of S2Lb-GFP on 40 μl samples or pooled as indicated before immunoprecipitation using a Crosslink IP kit (Thermo Scientific) and an anti-GFP antibody (ThermoFisher #A-11122).

For analysis of Tcb3 protein expression, 10 OD₆₀₀ units of Tcb3p-GFP–expressing cells post sterol depletion were prepared as described by Ohashi and colleagues [100 (link)]. Pellets were resuspended in SDS sample buffer and boiled for 5 min before SDS-PAGE. Protein transfer to nitrocellulose membranes and immunoblot conditions were as previously described [101 (link)]. To detect Tcb3p-GFP, immunoblots were incubated with a 1:1,000 anti-GFP antibody (ThermoFisher Scientific Inc., Waltham, MA) followed with 1:10,000 anti-rabbit-HRP secondary antibody (Bio-Rad Laboratories, Mississauga, ON). Actin was detected using 1:1,000 anti-actin antibody (Cedarlane, Burlington, ON) followed with 1:10,000 anti-mouse-HRP secondary antibody (ThermoFisher Scientific Inc.).
For analysis of Ysp2 protein expression, 10 OD₆₀₀ units of GFP-Ysp2–expressing cells were prepared and proteins extracted as above. To detect GFP-Ysp2, immunoblots were incubated with 1:2,000 anti-GFP antibody (Sigma-Aldrich Chemicals) followed by 1:10,000 anti-rabbit-HRP secondary antibody (Promega, Madison, WI). GAPDH was detected using 1:10,000 anti-GAPDH antibody (ThermoFisher Scientific Inc.) followed with 1:10,000 anti-mouse-HRP secondary antibody (Promega).

For western blot analysis, the proteins separated on SDS-PAGE gels were transferred onto a polyvinylidene fluoride membrane with a BioRad electroblotting apparatus as described [40 (link)]. The signals on the blots were detected with anti-p44/42 MAP kinase antibody (Cell Signaling Technology, MA) or anti-GFP antibody (Thermo Fisher Scientific, USA) under the ECL Supersignal system (Pierce, Rockford, IL).

Immunoelectron microscopy was performed as described in a previous study^{45 (link)} with some modifications. Arabidopsis root tips were frozen in a high-pressure freezing machine (EM PACT; Leica Microsystems, Wetzlar, Germany). The samples were freeze-substituted with 0.25% glutaraldehyde and 0.1% uranyl acetate in 100% acetone at −80 °C for 4 days, and then gradually warmed (EM AFS; Leica Microsystems). The samples were washed with 100% acetone, infiltrated with methanol, and then embedded in LR White resin (London Resin Company Ltd., London, UK). Ultrathin sections (70–80 nm) on formvar-coated nickel grids were labeled with anti-GFP antibody (1:50, A11122; Thermo Fisher Scientific, Waltham, MA, USA) in 50 mM Tris-buffered saline (TBS). After washing with TBS, sections were labeled with 12-nm colloidal gold particles coupled to goat anti-rabbit IgG (1:20, AB_2338016; Jackson ImmunoResearch, West Grove, PA, USA). The sections were stained with 4% uranyl acetate for 20 min and then examined under a transmission electron microscope (JEM-1400; JEOL, Tokyo, Japan) at 80 kV.

Cells were plated at 30,000 cells per well in 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific) 24 h prior to transduction. 24 h after plating, cells were transduced with AAVHSC7-226 at a MOI: 150,000. Before staining, cells were washed with PBS and fixed with 4% paraformaldehyde 48 h after transduction. They were then rinsed 3-times with PBS and blocked with PBS containing 3% BSA and 0.3% Triton X-100 for 1 h at room temperature. This was followed by incubation with primary anti-MeCP2 antibody (1: 100 dilution; Cell Signaling, #3456) and an anti-GFP antibody (1:500 dilution, Thermo Fisher, # MA5-15256) in PBS containing 1% BSA and 0.3% Triton X-100 at 4°C overnight. The cells were then rinsed 3-times with PBS and incubated with secondary antibodies. Secondary antibodies used were anti-rabbit Alexa Fluor-555 IgG (1:500, cell Signaling, #4413) and anti-mouse Alexa Fluor-488 IgG (1:500, cell Signaling, #4408) in PBS containing 1% BSA and 0.3% Triton X-100 for 2 h at room temperature in dark. The cells were finally rinsed 3-times with PBS, mounted with antifade reagent containing DAPI (VECTASHIELD Vibrance^®, #H-1800) and visualized at ×40 magnification using the Zeiss LSM 700 confocal microscope.