Promega
TnT SP6 High-Yield Wheat Germ Protein Expression System
L3260
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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, soluble proteins were expressed for in-vitro binding assay.
In-vitro protein binding assay
Constructs expressed from a pCS2+ backbone plasmid
Follow manufacturer's instructions.
1. Myc-tagged EFCAB7 full length and individual domain constructs were cloned into pCS2+ plasmid.2. Mix 6 µl High Yield WG mix (SP6), 2 µg plasmid DNA, 0.2 µl of 1mM Methionine (supplied with the kit) and nuclease-free water up to 10µl (supplied with the kit).3. Incubate at 25 deg C for 2 hours.4. After this point everything should be on ice or at 4 degrees.5. Add 1µl of 1mg/ml RNaseA to stop the reaction6. Briefly spin down tubes in a microcentrifuge and add proceed with in vitro binding assay.
10.1016/j.devcel.2014.01.021
Excellent system for soluble protein expression.
None.
Fast and easy soluble protein expression system for small scale applications.