Immunoprecipitation and Western Blot

IB assay was performed as previously described [27 (link)]. As to immunoprecipitation, cells were lysed, incubated with 2 μg corresponding antibodies overnight and incubated with protein A/G agarose for 4 h. The immunocomplexes were then resuspended with 2 × sample loading buffer and boiled for 5 min for SDS-PAGE and IB analysis. The primary antibodies used were: anti-Skp2 (ab68455, Abcam); anti-Skp1 (#12248, Cell Signaling Technology); anti-Cul1 (#4995, Cell Signaling Technology).

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Ma X., Zhao J., Yang F., Liu H, & Qi W. (2017). Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation. Oncotarget, 8(48), 84193-84203.

Publication 2017

ab68455 anti-Skp1 anti-Cul1

Agarose Antibodies Assay Buffer Cells Cul1 G protein Immunoprecipitation Protein a Protein g Sds page Skp1 Skp2

Corresponding Organization :

Other organizations : First Hospital of Jiaxing, Jiaxing University

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Variable analysis

independent variables

Antibodies used for immunoprecipitation (anti-Skp2, anti-Skp1, anti-Cul1)

dependent variables

Protein levels/interactions measured after immunoprecipitation and SDS-PAGE

control variables

Cell lysis method
Protein A/G agarose incubation time (4 h)
SDS-PAGE and IB analysis

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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