Protocol detail

CD3 Stimulation and GST-TSAd SH2 Pulldown

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The following mAb were used; anti-human CD3ϵ (OKT3, American Type Culture collection, Manassas, VA), anti-GST (B-14,Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphotyrosine (anti-pY) (clone 4G10, Upstate Biotechnology, Lake Placid, NY), and horseradish peroxidase-conjugated goat-anti mouse IgG mAb (Sigma-Aldrich). Pull-down experiments using anti-CD3 stimulated Jurkat E6.1 cells (American Type Culture Collection) and GST-TSAd SH2 fusion proteins on glutathione sepharose beads were performed as previously described [9 (link)].

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Andersen T.C., Lindsjø K., Hem C.D., Koll L., Kristiansen P.E., Skjeldal L., Andreotti A.H, & Spurkland A. (2014). Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO. BMC Biotechnology, 14, 3.

Publication 2014

anti-GST (B-14, Jurkat E6.1 cells

Anti cd3 Anti igg Clone Glutathione Goat Horseradish peroxidase Human Jurkat cells Mouse Phosphotyrosine Proteins Pull Sepharose

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Variable analysis

independent variables

Anti-human CD3ϵ (OKT3, American Type Culture collection, Manassas, VA)
Anti-GST (B-14,Santa Cruz Biotechnology, Santa Cruz, CA)
Anti-phosphotyrosine (anti-pY) (clone 4G10, Upstate Biotechnology, Lake Placid, NY)
GST-TSAd SH2 fusion proteins

dependent variables

Phosphorylation of proteins
Protein-protein interactions

control variables

Jurkat E6.1 cells (American Type Culture Collection)
Glutathione sepharose beads

positive controls

Anti-CD3 stimulated Jurkat E6.1 cells

negative controls

Not explicitly mentioned

Annotations

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