TCA-based Whole Cell Extraction and Immunoprecipitation

Whole cell extracts were prepared using the trichloroacetic acid (TCA) method as previously described (39 (link)). Immunoprecipitation was carried out following our published method (39 (link)). Proteins from whole-cell extracts or immunoprecipitated samples were resolved on a SDS-PAGE gel and transferred onto a PVDF membrane (Millipore) using a semi-dry transfer cell (Bio-Rad). Mouse monoclonal anti-HA antibody was purchased from Abcam, anti-Myc and anti-FLAG antibodies were purchased from Sigma, and the phosphor-specific rabbit polycolonal antibody against Fun30 phosphorylation on serine 28 was ordered from GenScript (Nanjing). Anti-mouse and rabbit IgG HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. Blots were developed using the ECL Prime Western Blotting substrate (GE Healthcare).

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Chen X., Niu H., Yu Y., Wang J., Zhu S., Zhou J., Papusha A., Cui D., Pan X., Kwon Y., Sung P, & Ira G. (2016). Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection. Nucleic Acids Research, 44(6), 2742-2753.

Publication 2016

PVDF membrane semi-dry transfer cell Mouse monoclonal anti-HA antibody anti-Myc and anti-FLAG antibodies Anti-mouse and rabbit IgG HRP-conjugated secondary antibodies ECL Prime Western Blotting substrate

Anti antibodies Antibodies Antibody Cell Cell extracts Igg hrp Immunoprecipitation Membrane Monoclonal antibody Mouse Phosphor Phosphorylation Proteins Pvdf Rabbit Sds page Serine Trichloroacetic acid

Corresponding Organization :

Other organizations : Wuhan University, Yale University, Baylor College of Medicine

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Variable analysis

independent variables

Whole cell extracts prepared using the trichloroacetic acid (TCA) method
Immunoprecipitation procedure

dependent variables

Protein expression and phosphorylation levels as detected by Western blotting

control variables

SDS-PAGE gel separation and PVDF membrane transfer
Antibodies used for detection (mouse monoclonal anti-HA, anti-Myc, anti-FLAG, and phosphor-specific rabbit polycolonal anti-Fun30 phosphorylation on serine 28)
HRP-conjugated secondary antibodies
ECL Prime Western Blotting substrate

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

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