Strep pull down assay

Transfect 10 ml HEK GNTi cells at concentration of 2 × 10^6 cells/ml

Dilute PEI with Warm Hybridoma-SFM(1X)

In a separate tube, dilute DNA with Hybridoma-SFM(1X)

Add PEI to DNA dilution. Incubate mixture for 0h 30m 0s at 37°C

Add mixture to cells. Let cells grow for 48h 0m 0s

Harvest Cells 500rpm,0h 0m 0s, 4°C, 0h 10m 0s

Wash pellet with cold PBS. Store pellet at-80°C until purification.

Homogenize the pellets in 0.5 ml of lysis buffer/protease inhibitors/1% TritonX-100, and clarified after 40000x g,0h 0m 0s , 0h 15m 0s.

Incubate the lysate with 30 μl Strep-Tactin Sepharose resin (IBA-Lifesciences) at 4°C for 3h 0m 0s

Wash the beads four times, and eluted in 50 μl lysis buffer/4 mM desthiobiotin.

Mix 18 μl eluent with lithium dodecylsulfate (LDS)/BME buffer, heated at 60°C for0h 5m 0s and subjected to SDS/PAGE gel. The gel was then stained with Coomassie brilliant blue G250.