The cells were lysed and the protein concentration was determined following the aforementioned procedure. Equal amounts of protein samples (160μg/80μl) were incubated with the anti-phosphotyrosine antibody (PY20, 1:500, Santa Cruz Biotechnology, Inc.), anti-phosphoserine antibody (p-Ser, 1:500, Santa Cruz Biotechnology, Inc.), and anti-phosphothreonine antibody (p-Thr, 1:500, Santa Cruz Biotechnology, Inc.) at 4°C for 1 hour respectively, followed by the addition of 15μl protein A/G plus-agarose beads (Santa Cruz Biotechnology, Inc.) and an incubation of 1 hour. The samples were washed with SDS-free lysis buffer twice before they were boiled with 1× sample buffer (Sigma). Proteins were probed with the anti-JNK antibody (1:500, Santa Cruz Biotechnology, Inc.), and anti-ERK antibody (1:500, Santa Cruz Biotechnology, Inc.), followed by a peroxidase-conjugated secondary antibody (1:2,000, Sigma). Protein bands were visualized using a chemiluminescence detection kit (Luminata, Millipore) and photographed using a Syngene imager (Syngene International Ltd.).
Anti phosphotyrosine antibody py20
Manufactured by Santa Cruz Biotechnology
Anti-phosphotyrosine antibody (PY20) is a mouse monoclonal antibody that recognizes phosphorylated tyrosine residues in proteins.
Automatically generated - may contain errors
2 protocols using anti phosphotyrosine antibody py20
1
Immunoprecipitation and Immunoblotting Analysis
2
In Vitro Lck-Mediated CD3ζ and PD-1 Phosphorylation
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Experiments were carried out as previously described (28 (link)). Briefly, proteins were purified using a baculovirus- or bacterial-expression system. Large unilamellar vesicles (LUVs) and proteins of interest were premixed and incubated at room temperature for 1 h. Then 2 mM ATP was injected and rapidly mixed to trigger Lck-mediated phosphorylation of CD3ζ and PD-1. Twenty minutes after ATP addition, apyrase was added (t = 0 min), and the reactions were allowed to continue at room temperature. Equal fractions of the reactions were removed and terminated with SDS sample buffer at the indicated time points. Anti-phosphotyrosine antibody (pY20, Santa Cruz Biotechnology; SC-508) was used to detect phosphorylation by Western blotting.
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