Rapid Immunoprecipitation for Transient Protein Interactions

Hsf1-3xFLAG-V5 was serially immunoprecipitated using anti-FLAG (M8823; Sigma-Aldrich) and anti-V5 (M167-11; MBL) magnetic beads, and eluates were blotted for IP/Western blot analysis (Zheng et al., 2016 (link); Zheng and Pincus, 2017 (link)). For IP/MS of Sis1-3xFLAG and Hsf1-3xFLAG, the protocol was modified to only perform the anti-FLAG IP using a short incubation of the anti-FLAG beads with total lysate for 15 min before washing and eluting with 3xFLAG peptide. The short incubation was designed to increase the likelihood of capturing transient interactions while reducing nonspecific interactions. All IP experiments were performed in biological triplicate. Triplicate IPs were also performed on an untagged strain exposed to the same conditions to subtract background and calculate significance. Immunoblots were performed with anti-FLAG (M2, F1804; Sigma-Aldrich), anti-V5 (V5-10, V8012; Sigma-Aldrich), or anti-Ssa1/2 (rabbit polyclonal; gift from Elizabeth A. Craig, Department of Biochemistry, University of Wisconsin, Madison, WI; Lopez-Buesa et al., 1998 (link)) antibodies as indicated.

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Feder Z.A., Ali A., Singh A., Krakowiak J., Zheng X., Bindokas V.P., Wolfgeher D., Kron S.J, & Pincus D. (2020). Subcellular localization of the J-protein Sis1 regulates the heat shock response. The Journal of Cell Biology, 220(1), e202005165.

Publication 2020

M8823

Antibodies Biological Immunoblots Peptide Rabbit Strain Transient Western blot analysis

Corresponding Organization : University of Chicago

Other organizations : University of Delaware, Henan Agricultural University

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Variable analysis

independent variables

Immunoprecipitation method (serial immunoprecipitation using anti-FLAG and anti-V5 magnetic beads vs. single anti-FLAG immunoprecipitation with short incubation)
Presence or absence of 3xFLAG tag on Hsf1 and Sis1

dependent variables

Protein-protein interactions detected by IP/Western blot analysis and IP/MS

control variables

Biological triplicate for each immunoprecipitation experiment
Untagged strain exposed to the same conditions as a negative control for background subtraction and significance calculation

controls

Positive control: Immunoblots performed with anti-FLAG, anti-V5, and anti-Ssa1/2 antibodies
Negative control: Untagged strain exposed to the same conditions

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