Vector Laboratories
Streptavidin Agarose Ultra Performance
N-1000-002
Info: Supplier Page
Hedgehog signaling plays a critical role in embryonic development. It also controls adult tissue homeostasis and deregulation of this pathway results in basal cell carcinoma and medulloblastoma. A unique feature of this pathway is that it requires a tiny antenna-like organelle called the primary cilium for signal transduction. My goal is to understand the molecular mechanism of hedgehog signal transduction at the primary cilium. This involves detecting protein-protein interactions of the pathway components, analysis of localization patterns during signal transduction, and transcriptional output measurements. In this experiment, plasma membrane enriched Patched 1 (the receptor for the hedgehog pathway) was analyzed in NIH-3T3 cells.
Pull down assay
Cell surface biotinylated proteins from NIH-3T3 cells
2 hrs incubation with the resin at 4 deg C.
Pull down assay was set up with 400 µg lysate and 10 µl resin (in 500 µl volume) for 2 hrs at 4 °C on a rotator. Beads were washed extensively and biotinylated proteins were eluted by adding 1x NuPAGE LDS Sample Buffer (containing 100 mM DTT that is suitable for the biotinylation reagent in this experiment) and incubating at 37 °C for 1 h. Samples were resolved on a 4-12% Bis-Tris Gel, proteins were transferred onto nitrocellulose membrane, and blotted for Patched 1 antibody.
N/A
5-10 µL of resin is more than enough for small scale streptavidin pull down assays.
None.
Good streptavidin agarose resin with high binding capacity!