Rabbit polyclonal anti snap tag

For Western blot analysis, membranes were blocked with 5% milk in TBST for 1 hour at room temperature. Primary antibody incubation was carried out in 5% milk in TBST either for 2 hours at room temperature or for 14 hours at 4°C. Secondary antibody incubation was performed in 5% milk in TBST for 1 hour at room temperature. Primary antibodies used were: Mouse monoclonal anti-LIS1 (Cat no. sc-374586, Santa Cruz Biotechnology, 1:1000 dilution), Mouse monoclonal anti-Dynein IC1/2 (Cat no. sc-13524, Santa Cruz Biotechnology, 1:1000 dilution), Mouse monoclonal anti-GAPDH (Cat no. ab8245, Abcam, 1:1000 dilution), Rabbit polyclonal anti-SNAP-tag (Cat no. P9310S, New England Biolabs, 1:1000 dilution). Secondary antibodies used were: Goat anti-mouse IgG HRP (Cat no. P0447, Dako, 1:1500 dilution) and Goat anti-rabbit IgG HRP (Cat no. ab6721, Abcam, 1:2000 dilution).

The same PEG-coated quartz slides were used for the single-molecule IP assays. All samples were diluted in and washed with H50N50 buffer. In total, 0.1 mg/mL NeutrAvidin was added to and incubated first on a quartz slide for 3 min before being removed by washing with H50N50 buffer. In all, 5 μg/mL secondary antibody (i.e., biotinylated goat anti-mouse or rabbit IgG antibody) and 5 μg/mL primary antibody (i.e., mouse monoclonal Anti-NSF (Santa Cruz Biotechnology) or rabbit polyclonal Anti-SNAP tag (New England Biolabs)) were added and incubated for 5 min. After removing the free antibodies by washing, 1 nM NSF for anti-N-domain or 20 pM NSF for anti-SNAP tag with 1 mM ATP and 1 mM EDTA was added and incubated for 5 min. The free proteins were removed by washing with H50N50 buffer containing 1 mM ATP/1 mM EDTA. An oxygen scavenger system (2.5 mM PCA, 0.5 unit/ml PCD, 1 mM Trolox) with 1 mM ATP/1 mM EDTA was injected onto the slide, and fluorescence time traces were collected at a frame rate of 10 Hz for about 400 s.

Primary antibodies used in this study are as listed: mouse monoclonal anti-Halo (Promega, G9211), rabbit polyclonal anti-SNAP-tag (New England BioLabs, P9310S), mouse monoclonal anti-HSP90 (BD Transduction Laboratories, 610419), anti-β-actin (Sigma-Aldrich, A2228), rabbit polyclonal anti-LC3 (Kabeya et al., 2000 (link)), rabbit polyclonal anti-GFP (Thermo Fisher Scientific, A6455), and mouse monoclonal anti-RFP (MBL, M155-3). Secondary antibodies are HRP-conjugated goat polyclonal anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-144) and HRP-conjugated goat polyclonal anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-035-003). LysoTracker Red DND-99 or Deep Red (Thermo Fisher Scientific, L7528, L12492) were applied at 75 nM.