Human/Mouse NLRP3/NALP3 Antibody

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	-Rat (1:250)

Protocol tips
-Rat (1:250)

Publication protocol

Total proteins of CD4+ T cell samples were extracted in Laemmli buffer (Tris-HCl 62.5 mmol/l, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol), and their concentration was measured. Thirty-five micrograms of total protein was electrophoresed on 7% SDS-PAGE gels and blotted onto nitrocellulose (10401196, Schleicher & Schuell). Blots were then stained with Ponceau S. Membranes were blocked for 1 hour in TBS (Tris 10 mmol/l, NaCl 150 mmol/l), 0.1% Tween-20, 5% nonfat dry milk, pH 7.4 at 25°C, incubated for 12 hours with rat anti-NLRP3 (MAB7578, R&D Systems) diluted 1:250 or with rabbit anti-P2X7R (P8232, Sigma-Aldrich) diluted 1:200 in TBS–5% milk at 4°C, washed 4 times with TBS–0.1% Tween-20, then incubated with goat anti-rat–HRP (sc-2006, Santa Cruz Biotechnology) or goat anti-rabbit–HRP (P0448, Agilent Technologies) diluted 1:500 in TBS–5% milk, and finally washed with TBS–0.1% Tween-20. The resulting bands were visualized using enhanced chemiluminescence (32132, SuperSignal, Pierce Biotechnology Inc.).

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