Anti-Smad2 (phospho S467) antibody (ab53100)

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

Cells were lysed by sonicating for 5 s in 1 ml of detergent-free lysis buffer (PBS, 5 mM EDTA, 0.02% sodium azide, 10 mM iodoacetamide, 1 mM PMSF, and 2 mg leupeptin) at 4°C. Antibody-conjugated beads were prepared by combining 1 mg of antibodies with 30 ml of a 50% protein A Sepharose bead slurry in 0.5 ml of ice-cold PBS for 1 hr at 4°C in a tube rotator and were then washed three times with 1 ml of lysis buffer. The antibodies used for coimmunoprecipitation were Akt (ab6076, abcam). The beads were washed three times with washing buffer (50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, 0.02% sodium azide, 0.1% Triton X-100) and once with ice-cold PBS. Immunodetection was carried out using the ECL Western Blotting Detection Kit (Amersham Corp). Proteins were detected using antibodies against Smad4 (ab40759, abcam), Pitx2 (sc-8748, Santa Cruz), p-Smad3 (ab52903, abcam), p-Smad2 (ab53100, abcam), B55α (ab185712, abcam), pAKT-T308 (ab5626, abcam), pAKT-S473 (ab8932, abcam) and GAPDH (ab9485, abcam). Relative protein expression levels were normalized to GAPDH levels.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Smad2 (phospho S467) antibody (ab53100) below.

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