Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Publication protocol

1 × 106 cells were washed twice with PBS and lysed in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40 (Calbiochem, 492015), 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid (SIGMA, D-6750), 0.1% SDS (SERVA, 39575.02) 1% Triton X-100, protease and phosphatase inhibitors (SIGMA, S8830, S6508 and 450022) The lysates were subjected to electrophoresis on 4–12% NuPage Bis-Tris gels (Life Technologies/Novex, NP0323) and transferred to nitrocellulose membranes (GE Healthcare/Amersham, 10600002 Protran). The membranes were then blocked in 1X PBS-0.1% Tween 20 (SIGMA, P1379) containing 3% BSA (SIGMA, A4503) and probed with specific primary antibodies, overnight at 4°C. After several washes in 1X PBS-0.1% Tween 20 the membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, sc-2004, sc-2005 and sc-2768), used at 1:10,000. Finally, the membranes were washed in 1X PBS-0.1% Tween 20 and immunoreactivity was detected using an enhanced chemiluminescence kit (Thermo Scientific, 32209). Densitometric analysis was performed using ImageJ software.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb below.

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