Phosphorylation Detection of Gad8 and Chk1

Proteins were resolved by SDS-PAGE 10–15% acrylamide gels and transferred to nitrocellulose membranes, blocked with 5% milk in TBST and immunoblotted with the indicated antibodies. Detection was carried out using the ECL SuperSignal detection system (Thermo Scientific). Gad8 Thr387 phosphorylation events were detected using total protein extracts by phosphospecific antibodies. Antibodies against Gad8 Thr387-P were raised against the Gad8 phosphopeptide CRFANWpSYQRPT [59 (link)]. To follow Chk1 phosphorylation, proteins were extracted with trichloroacetic acid (TCA) as described previously and resolved by SDS-PAGE using 8% acrylamide gels.

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Pataki E., Simhaev L., Engel H., Cohen A., Kupiec M, & Weisman R. (2020). TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast. PLoS Genetics, 16(11), e1009196.

Publication 2020

ECL SuperSignal detection system

Acrylamide Antibodies Gels Membranes Milk Nitrocellulose Phosphopeptide Phosphorylation Phosphospecific antibodies Proteins Sds page Trichloroacetic acid

Corresponding Organization : Tel Aviv University

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Variable analysis

independent variables

Protein samples resolved by SDS-PAGE 10–15% acrylamide gels
Protein samples transferred to nitrocellulose membranes
Protein samples blocked with 5% milk in TBST
Protein samples immunoblotted with indicated antibodies
Protein samples extracted with trichloroacetic acid (TCA) and resolved by SDS-PAGE using 8% acrylamide gels

dependent variables

Gad8 Thr387 phosphorylation events detected using phosphospecific antibodies
Chk1 phosphorylation detected

control variables

ECL SuperSignal detection system (Thermo Scientific) used for detection

positive controls

Antibodies against Gad8 Thr387-P raised against the Gad8 phosphopeptide CRFANWpSYQRPT

negative controls

No negative controls explicitly mentioned

Annotations

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