LC3B Antibody

Protocol tips

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	For WB dilute primary Ab at 1:1000 and for IF at 1:200 and incubate at 4°C with gentle shaking, overnight.

Protocol tips
For WB dilute primary Ab at 1:1000 and for IF at 1:200 and incubate at 4°C with gentle shaking, overnight.

Publication protocol

Western blot, immunoprecipitation, and Phos-tag gel electrophoresis
For western blotting, equal amounts of protein from each lysate were loaded on polyacrylamide gels and after separation by gel electrophoresis blotted onto nitrocellulose membranes. Membranes were incubated overnight with primary antibody, washed 3 times with washing buffer (Tris-buffered saline [0.2 M Tris base (Sigma-Aldrich, 10708976001) 1.5 M NaCl (Sigma-Aldrich, S3014)] 0.05% Tween (Sigma-Aldrich, P1379), 3% milk powder (Roth, T145.3) before incubation with horseradish peroxidase-coupled anti-mouse, anti-rabbit or anti-goat secondary antibodies (Santa Cruz Biotechnology, sc-2005, sc-2030, and sc-2020, respectively) for 2 h. Immunoreactive proteins were detected with a Fusion Solo S imager (Vilber Lourmat) using an enhanced chemiluminescence detection kit (Witec AG, K12045).

For immunoprecipitation, samples were pre-cleared with Sepharose G beads (GE Healthcare, 17–0618–01) and incubated on a rocker overnight at 4°C with 10 μg/ml anti-PTPN22, 10 μg/ml anti-mouse NLRP3, 15 μg/ml anti-human NLRP3 or 7 μg/ml anti-mouse/human SQSTM1 (Santa Cruz Biotechnology, sc-25575) antibodies, before precipitation with Sepharose G beads. After washing, the pellet was resuspended in 1x loading buffer, boiled for 10 min at 95°C, and the supernatants loaded on polyacrylamide gels. For detection of precipitated proteins, the same procedure was applied as used for western blotting.

To discriminate phosphorylated from nonphosphorylated proteins, Phos-tag gels were obtained from Wako Chemicals (304–93–521), and used according to the manufacturer's instructions and as described previously.39 In brief, cell lysates or autophagosome-enriched cell fractions were separated through 12% polyacrylamide gels containing 50 μM MnCl2 and 25 μM Phos-tag ligand. Gels were then blotted on nitrocellulose membranes and proteins detected using the same methods as for western blotting.

Confocal microscopy
For confocal microscopy, THP-1 cells were differentiated into macrophages on cover slides using 100 μg/ml PMA (Sigma-Aldrich, P1585) for 48 h, before activation with MSU for 6 h. Antibodies used for detection of LC3B and NLRP3 were the same as described above, goat anti-mouse Alexa Fluor 647 (ThermoFisher Scientific; A-21235) and goat anti-rabbit Alexa Fluor 564 (ThermoFisher Scientific; A-11035) were used as secondary antibodies. Immunofluorescence confocal microscopy was performed using a CSLM SP5 Leica microscope and image analyses performed using the LAS lite imaging software.

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