Recombinant protein production and pull-downs
This protocol is extracted from research article:
KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation
Sci Signal, Jan 30, 2018; DOI: 10.1126/scisignal.aaq1060

The cDNAs encoding the GST-NT-KIF22 and GST-CT-KIF22 domains were provided by A. Wilde (University of Toronto). BL21 cells containing DNA of interest were grown in 400 ml of LB broth at 37°C and then protein production was induced with isopropyl β-d-1-thiogalactopyranoside (100 μM) and incubated for a further 4 hours at 30°C. The bacterial culture was pelleted and frozen overnight at −80°C and then lysed in 50 μl of ice-cold PBS per milliliter of original culture volume containing protease inhibitors (Calbiochem). Bacterial pellets were disrupted by sonication for 2 min using 10-s pulses at 10 A on ice. The solution was centrifuged, and the supernatant was collected and filtered using a 0.45-μm filter. Prewashed glutathione Sepharose 4B beads (GE Healthcare) were added to the supernatant to 1 μl of beads/1 ml of original culture and left to mix overnight at 4°C. The beads were washed twice with PBS containing 0.4 M NaCl and 1% Brij and resuspended in 1 ml of buffer with protease inhibitors. SDS-PAGE and Coomassie blue analysis were run to confirm protein purification.

A549 or HEK293T cells were washed and scraped into cold lysis buffer [50 mM tris (pH 7.2), 150 mM NaCl, 20 mM EDTA, 1% NP-40, and 1% Triton X-100] containing protease inhibitor cocktail and phosphatase inhibitors (100 μM vanandate and 1 μM calyculin A). Lysates were then centrifuged to pellet insoluble material. A sample of cell lysate was removed before adding lysate to beads to allow analysis of total protein. The cleared lysates were transferred into tubes with 50 μl of GST-tagged protein and placed on a rotator at 4°C overnight. The beads were washed four times with ice-cold lysis buffer. After the final wash, the wash buffer was removed and SDS sample buffer was added. The sample was boiled and run on a 12% acrylamide gel and analyzed by immunoblotting.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.