Anti-activated Notch1 antibody (ab8925)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:500) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Tissues were harvested in lysis buffer (10 mM Tris at pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) with protease and phosphatase inhibitor cocktail (Thermo Scientific). The lysates were briefly sonicated and centrifuged at 14,000 rpm for 20 min at 4°C. Protein concentrations were determined using the Bio-Rad protein assay. Samples were then mixed with Laemmli buffer (2% SDS) and boiled for 5 min. Proteins were resolved in SDS–polyacrylamide gels, transferred to nitrocellulose, and incubated with one of the following primary antibodies: APC (1:500; Santa Cruz Biotechnology, C-20), β-actin (1:1000; Abcam, ab8226), β-catenin (1:1000; Sigma, C2206), Gli1 (1:500; Cell Signaling, 2534), Gli2 and Gli3 (1:200; gift from Dr. B. Wang, Cornell University), Hes1 (gift from Dr. R. Kageyama, Kyoto University; 1:500), NICD (1:500; Abcam, ab8925), Notch1 (1:500; Santa Cruz Biotechnology, C-20), p21 (1:200; Santa Cruz Biotechnology, F-5), or RBP-jk (1:1000; Millipore, AB2284). Immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies and detected with enhanced chemiluminescence.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-activated Notch1 antibody (ab8925) below.

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