Procedure - remove GST from hepsin to prepare for pulldown:<br />1. Dilute GST-Hepsin by bringing up to 10mL 50mL Tris-HCl<br />2. Use centriprep concentrator (10kDa MW cutoff) to concentrate back to small volume (<500 uL)<br />Procedure - preclear cell lysate<br />1. Add cell lysate to 75 uL unlabeled sepharose beads and mix end to end at 4oC 1h. I used ~30ug cell lysate.<br />2. Pellet beads and preserve supernatant<br />Procedure - prepare Hepsin beads<br />1. Use GE healthcare Sepharose 4B beads. Prepare according to GE Healthcare Batch Purification Method. Use 40uL beads per sample.<br />2. Add 1ug Hepsin to resin, mix end to end 1h at room temp<br />3. Wash with 200uL PBS<br />Procedure - pulldown<br />1. Add pre-cleared cell lysate and mix end to end overnight at 4C<br />2. Wash 3x with PBS<br />3. Add 150ug glutathione in 50mM Tris pH8 and mix at room temp for 30 min and at 4C for 30 min<br />4. Pellet the agarose and use the supernatant for western blotting (see image. All constructs were HA tagged and the western blot used an anti-HA primary and HRP-secondary)
5% Milk in TBST, 1h at RT
Cell Signaling Technologies Rabbit anti-HA 1/1000 O/N at 4 degrees in blocking buffer
negative control: flag tagged construct
Sepharose 4B GST GE Healthcare. See image or comments for full protocol
See image or comments for protocol.
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