Immunoprecipitation and Immunoblotting of HA-Abl

Transfection of HEK 293 cells and immunoprecipitation of HA-tagged Abl protein was carried out as described previously [5] (link), [7] (link) using the monoclonal Anti-HA agarose conjugate (Clone HA-7, Sigma-Aldrich). Immunoblotting was done using the following antibodies: anti-phosphotyrosine (4G10, Millipore), anti-phospho-Abl (Tyr412, Cell Signaling Technology), and anti-HA labeled with AlexaFluor 680 (Molecular Probes). Secondary antibody was goat anti-mouse IgG labeled with AlexaFluor 680 (Molecular Probes). Alternatively, peroxidase-labeled mouse anti-HA antibody (HA-7, Sigma) was used. Quantification of the relative amounts of immunoprecipitated Abl protein was done using the Odyssey system (Li-Cor) or ImageJ program.

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Dölker N., Górna M.W., Sutto L., Torralba A.S., Superti-Furga G, & Gervasio F.L. (2014). The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion. PLoS Computational Biology, 10(10), e1003863.

Publication 2014

Anti-HA agarose conjugate (Clone HA-7, anti-phosphotyrosine (4G10, AlexaFluor 680 Odyssey system

Agarose Anti antibody Anti igg Antibodies Antibody Clone Goat Hek 293 cells Immunoprecipitation Molecular probes Mouse Peroxidase Phosphotyrosine Protein Transfection

Corresponding Organization : University College London

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Variable analysis

independent variables

Transfection of HEK 293 cells
Immunoprecipitation of HA-tagged Abl protein

dependent variables

Relative amounts of immunoprecipitated Abl protein

control variables

Monoclonal Anti-HA agarose conjugate (Clone HA-7, Sigma-Aldrich)
Anti-phosphotyrosine (4G10, Millipore) antibody
Anti-phospho-Abl (Tyr412, Cell Signaling Technology) antibody
Anti-HA labeled with AlexaFluor 680 (Molecular Probes) antibody
Goat anti-mouse IgG labeled with AlexaFluor 680 (Molecular Probes) secondary antibody
Peroxidase-labeled mouse anti-HA antibody (HA-7, Sigma)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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