Immunoprecipitation and Western Blotting of Proteins

Human embryonic kidney 293T cells (CRL-3216; American Type Culture Collection) were cultured in poly-L-lysine coated 6-cm plates (46 (link)). Upon reaching 25% confluency, cells were transiently transfected by the calcium phosphate coprecipitation method (47 (link)). Two days after transfection, cell extracts were prepared (48 (link)) and immunoprecipitations performed as previously described (49 (link)). Precipitated proteins were run on SDS polyacrylamide gels (50 (link)), transferred to polyvinylidene fluoride membrane (51 (link)), and then challenged with indicated antibodies (52 (link)). After incubation with corresponding secondary antibodies coupled to horseradish peroxidase (53 ), signals were revealed by chemiluminescence (54 (link)) and exposure to film (55 (link)). Likewise, endogenous proteins were coimmunoprecipitated from human HCT116 colorectal cancer cells, utilizing control rabbit IgG (Santa Cruz, sc-2027) or rabbit polyclonal anti-JMJD1A antibodies (Bethyl, A301-538A) for immunoprecipitation followed by Western blotting with anti-JMJD2A antibodies (Bethyl, A300-861A).

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Sui Y., Jiang H., Kellogg C.M., Oh S, & Janknecht R. (2023). Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7. Frontiers in Oncology, 13, 1122238.

Publication 2023

293t cells A301 Anti antibodies Antibodies Calcium phosphate Cell extracts Cells Chemiluminescence Colorectal cancer Embryonic Hct116 cells Horseradish peroxidase Human Immunoprecipitation Kidney Lysine Membrane Poly Polyacrylamide gels Polyvinylidene fluoride Proteins Rabbit Transfection

Corresponding Organization : Oklahoma State University Oklahoma City

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Variable analysis

independent variables

Transfection method (calcium phosphate coprecipitation)

dependent variables

Protein expression and interactions (measured by immunoprecipitation, SDS-PAGE, Western blotting, and chemiluminescence)

control variables

Cell line (Human embryonic kidney 293T cells, CRL-3216)
Cell culture conditions (poly-L-lysine coated 6-cm plates, 25% confluency)
Antibodies used for immunoprecipitation and Western blotting
Positive control: Endogenous proteins coimmunoprecipitated from human HCT116 colorectal cancer cells using anti-JMJD1A antibodies
Negative control: Endogenous proteins coimmunoprecipitated from human HCT116 colorectal cancer cells using control rabbit IgG

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