Mouse anti his tag antibody

For sample analysis, 5 µL of PageRuler^TM Plus Prestained Protein Ladder (Thermo Fisher Scientific, Vilnius, Lithuania) and 500 ng of low and highly phosphorylated Tau proteins were loaded for each condition on a 4%–20% polyacrylamide gel (Mini-PROTEAN^® TGX™, Bio-Rad, Hercules, CA, USA), and allowed to migrate during 1 h at 50 mA in 1× Tris-Glycine-SDS buffer (Bio-Rad). Boiling and β-mercaptoethanol were avoided to preserve multimeric complexes. The transfer was performed at 4 °C for 1 h 45 min and 260 mA (1× Tris-Glycine buffer (Bio-Rad) with 20% methanol) on a 0.45 µm nitrocellulose membrane (Amersham^TM Proton^TM, GE Healthcare, Berlin, Germany) which was incubated overnight with the corresponding primary antibodies diluted in Tris buffered saline (TBS)-Tween-20 0.05% (Sigma-Aldrich, St Louis, MO, USA) containing 5% bovine serum albumin (BSA): rabbit anti-phosphoT231 antibody 1:1000 (Abcam, Cambridge, UK), mouse anti-Tau antibody 1:1000 (Invitrogen, Carlsbad, CA, USA) and mouse anti-His-tag antibody 1:2000 (Invitrogen). Incubation with secondary antibodies was carried out for 1 h, either with an anti-mouse or an anti-rabbit (1:10,000) from sheep coupled to horseradish peroxidase (HRP) (GE Healthcare Biosciences, Uppsala, Sweden) and diluted in TBS-Tween-20 0.05%. All washing steps were accomplished in TBS-Tween-20 0.05%.

Colocalization of GPI-m36.4 or GPI-FluIgG03 with a lipid raft marker (GM1) was evaluated as described previously^{38 (link),43 (link)}. In brief, transduced TZM-bl cells were seeded (8000 cells/well) in a 35-mm glass dish with a 14-mm bottom well (Cellvis) and incubated for 2 days at 37 °C in 5% CO₂. After two washes with PBS, the cells were fixed with 4% formaldehyde in PBS containing 1% BSA for 15 min and blocked with blocking buffer (5% goat serum in PBS containing 1% BSA) for 1 h. Then, the cells were sequentially stained with a mouse anti-His tag antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG antibody, and Alexa Fluor 555-conjugated CtxB (Invitrogen Life Technologies). After three washes with PBS, the cells were further stained with DAPI in permeabilization buffer (blocking buffer plus 0.5% saponin) for 7 min. Images were captured with a laser confocal microscope (Leica Microsystems, Wetzlar, Germany).

Colocalization of GPI-anchored transgenes with a lipid raft marker (GM1) was determined by confocal microscopy as described previously [26 (link),27 ]. Briefly, transduced TZM-bl cells were seeded in a 35-mm glass dish with a 14-mm bottom well (Cellvis) at 8,000 cells per well and incubated for 2 days at 37°C in 5% CO₂. After two washes with PBS, the cells were fixed with 4% formaldehyde in PBS containing 1% BSA for 15 min and blocked with blocking buffer (5% goat serum in PBS containing 1% BSA) for 1 h. Subsequently, the cells were sequentially stained with a mouse anti-His tag antibody, Alexa Fluor 488- conjugated goat anti-mouse IgG antibody, and Alexa Fluor 555- conjugated CtxB (Invitrogen Life Technologies). The cells were washed with PBS three times and further stained with 4′,6-diamidino-2-phenylindole (DAPI) in permeabilization buffer (blocking buffer plus 0.5% saponin) for 7 min. Images were captured with a laser confocal microscope (Leica Microsystems, Wetzlar, Germany).

Samples of Rab3A heterologous expression and purification were resolved on a 10 % SDS-PAGE gel in principle as described by Laemmli (1970 (link)), followed by visualization with Coomassie brilliant blue staining and scanning with a G:BOX Gel imaging system (Syngene, Cambridge, UK). For further identification of the expressed Rab3A fusion protein, the protein in the corresponding band was transferred from gel lane onto a nitrocellulose membrane (PALL Corporation, USA) using a blot electrotransfer apparatus in the wet transfer method (100 mA/2.5 h) and blocked in 5 % milk/TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1 % Tween-20, pH 7.5) for 1.5 h at room temperature, and then probed with the mouse anti-His tag antibody (Novex, Life Technology, USA) (1:5000 dilution in 5 % milk/TBST) for 1.5 h at room temperature. After the membrane was washed three times (each for 6 min) using TBST, it was incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Promega Corporation, Madison, WI, USA) (1:8000 dilution in 5 % milk/TBST), followed by washing with TBST extensively. The blot was developed using the enhanced chemiluminescence (ECL) method (Thermo Scientific, USA) and recorded with a ChemiDoc XRS imaging system (Bio-Rad, USA).

ELISA was used to determine the half-life of the conjugates circulating in vivo. BALB/c mice (female, 7 weeks old) were injected with the conjugates at a dosage of 5.0 mg/kg per mouse via the tail vein. Blood samples were collected from the eye vein at different time points (3, 60, 120, 240, 360, 600, and 1440 min). The plasma was kept at room temperature for 30 min and then centrifuged at 4 °C to separate the serum. Human HER2 protein was coated on microtiter plates (5.0 mg/mL) and incubated at 4 °C for 18 h. The plates were washed with 0.1% PBS-T (PBS with 0.1% Tween-20) and blocked with 3% BSA solution for 1 h before adding serum samples diluted 1:400 with PBS buffer at 100 μL/well. After incubation for 1 h, the plates were washed with PBS-T again, incubated with mouse anti His-Tag antibody (1:2000 dilution, catalog No. 37-2900; Life Technologies, Camarillo, CA, USA) at room temperature for 1 h and with HRP-labeled goat anti-mouse IgG (1:2000 dilution, catalog No. CW0102; CWBio, Beijing, China) at room temperature for another 1 h. After washing with PBS-T, the plate was incubated with TMB for 15 min. The reaction was stopped with 100 μL 2.0 N sulfuric acid, and the absorbance was measured at 450 nm. The solution containing quantitative protein was used to draw the standard curve. GraphPad Prism 6.0 software was used to calculate the half-life of the conjugates.

Lysates from adult heads homogenized and sonicated in 2x Laemmli, cell lysates or immunoprecipitated protein complexes were separated by SDS-PAGE and transferred to PVDF membranes. Blots were incubated overnight at 4°C with the following antibodies: mouse anti-SGG (1:500; clone 4G-1E, Millipore), mouse anti-SGG (1:500, clone 7G1F2, Papadopoulou et al., 2004 (link)), mouse anti-phospho-S9-SGG (1:250; clone 7G2G5, Papadopoulou et al., 2004 (link)), mouse anti-phospho-Y214-SGG (1:250; clone 5G2F12, Papadopoulou et al., 2004 (link)), rabbit anti-GAPDH (1:500, Novus Biologicals, Littleton, CO, United States), rabbit anti-PER (1:10000, Stanewsky et al., 1997 (link)), mouse anti-Myc (1:1000, clone 9E10, Santa Cruz Biotech.), mouse anti-His-tag antibody (1:2000, clone HIS.H8, Thermo Fisher Scientific), mouse anti-α-Tubulin (1:5000, clone NDM1A, Merck, Darmstadt, DE). After incubation with HRP-coupled secondary antibodies, signal detection was done with the ECL Plus detection reagents (GE Healthcare Life Science) and a ChemoCam ECL Imager equipped with a 16bit camera (Intas, Göttingen, DE). Exposure times were adjusted to allow for quantification of signal intensities within the dynamic range of the camera system.