Anti-JAK2 antibody (ab39636)

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

The renal tissue was lysed in ice-cold radioimmunoprecipitation assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, and 0.1 mM phenylmethylsulfonyl fluoride] to obtain the total proteins. The protein concentration was detected using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein samples (50 µg protein/lane) were separated by 10–12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The non-specific antibodies were blocked with 5% non-fat dried milk in phosphate-buffered saline for 2 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies directed against JAK2 (cat. no. ab39636; 1:1,000; Abcam, Cambridge, UK), p-JAK2 (cat. no. ab32101; 1:500, Abcam), STAT3 (cat. no. ab68153; 1:1,000; Abcam), p-STAT3 (cat. no. ab76351; 1:500; Abcam), p65 (cat. no. ab16502; 1:1,000; Abcam) and p-p65 (cat. no. ab86299; 1:500; Abcam). The membranes were then washed with Tris-buffered saline with Tween 20 three times and further incubated with horseradish peroxidase conjugated secondary antibody (1:3,000; Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA) for 1 h at room temperature. Following washing, the membranes were processed using an electrochemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburg, PA, USA) and the light emission was captured on X-ray film. The signals were visualized by chemiluminescent horseradish peroxidase substrate and then subjected to a densitometric analysis and normalized to β-actin (1:3,000; Abmart Co, Ltd., Shanghai, China).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-JAK2 antibody (ab39636) below.

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