Thermo Fisher Scientific
Dynabeads Protein G for Immunoprecipitation
10004D
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The WNT pathway is essential for embryonic development and adult tissue homeostasis. Deregulation of this pathway results in cancers of the stomach, intestine, breast, etc. I am interested in studying the molecular mechanism of oncogenic WNT signal transduction to identify potential targets for therapeutic intervention of colon cancer. The goal of this experiment is to immunoprecipitate endogenous beta-catenin using an antibody and Dynabeads Protein A.
Immunoprecipitation
RKO nuclear extracts
IP was done at 4 deg C for 16 hr.
RKO (human colorectal cancer cell line) cells were treated with or without WNT3A conditioned medium for 4 hr. Nuclear extracts were prepared and 600 µg of extract was incubated with 1 µg of beta-catenin-10 µL Dynabeads Protein G bead complex overnight at 4 deg C. Beads were washed five times with lysis buffer and immune complexes were eluted in 2x SDS sample buffer (95 deg C heating for 5 min). Proteins were resolved on a 4-12% Bis-Tris gel and transferred to a nitrocellulose membrane.
N/A
Washing steps are relatively easy with magnetic beads.
None.
Excellent antibody capture reagent for immunoprecipitation application.