Anti-IKK beta (phospho Y199) antibody (ab59195)

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 (1:1000) -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 (1:1000) -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

Cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS and EDTA. Protein concentrations were measured using an Enhanced BCA Protein Assay Kit (Beyotime). Cytoplasmic and Nuclear Protein Extraction kits (Boster, Wuhan, China) were used to prepare nuclear and cytoplasmic extracts. Equal amounts of proteins (50 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk (diluted in TBST) for 2 h, and then incubated overnight with specific primary antibodies. After washing with TBST containing 0.1% Tween 20, the membranes were subsequently incubated with ECL horseradish peroxidase-linked secondary antibodies. The PVDF membranes were exposed to film, and the protein bands were visualized using a scanner system and densitometric analysis with Bandscan software. The relative densitometric ratio of the experimental bands was calculated by normalization using a loading control (LaminB1 or β-actin).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-IKK beta (phospho Y199) antibody (ab59195) below.

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