Recombinant Anti-STAT3 (phospho S727) antibody [E121-31] (ab32143)

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 (1:1200) -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 (1:1200) -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

HepG2 cells (1 × 105) treated with recombinant human IL-17, STAT3 inhibitor S31-201 or IL-6 mAb were cultured for 24 h. Total protein was extracted from the cells and samples, and the protein concentration was determined by the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, Waltham, MA, USA). Equal levels of protein were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and subsequently transferred to nitrocellulose membranes; then, the membranes were blocked with 5% nonfat milk for 2 h at room temperature and incubated overnight at 4°C with primary antibodies for STAT3 (ab68153, Abcam, UK, 1: 900), p-STAT3 (E121-31, Abcam, 1: 1200) and β-actin (ab8227, Abcam, 1: 700); β-actin was used for normalization. Then, incubation with secondary antibodies (Beyotime Biotechnology, Shanghai, China) for another 2 h was followed by washing with phosphate buffer (PBS). The signals were detected using the enhanced chemiluminescence substrate kit (Thermo Fisher Scientific). Protein bands were semi-quantified by ImageJ software.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-STAT3 (phospho S727) antibody [E121-31] (ab32143) below.

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