Bioanalytical Research Group

iTAP Based Protein Complex Purification

Generation of stable cell lines


In a six-well dish, seed 2-5 x 106 Schneider cells. Let adhere for at least 1 hour.

Transfect with a mixture of the plasmid pBSactTAP and the plasmid carrying the selection marker, pBS-PURO (Puromycin Acetyl Transferase). Use a ratio of 5:1, i.e. 2 µg plasmid pBSactTAP and 0.4 µg pBS-PURO per well. This ratio can be increased up to 15:1. Lipofectin protocol:

Prepare in sterile 1.5 ml reaction tubes the following mixtures: 

0.4 µg pBS-PURO and 2 µg plasmid pBSactTAP in 100 µl serum free medium

15 µl Lipofectin in 100 µl serum free medium. (Shake Lipofectin gently if it has not been used for a long time.) 

Allow A and B to stand at room temperature for 45 min, then combine A and B, mix gently, wait another 15 min; in the meanwhile: wash cells twice with serum free medium

Add 800 µl serum free medium to the tubes containing the Lipofectin-DNA complexes, mix gently and overlay the complex on the washed cells.

After 24 hours: Exchange Lipofectin/DNA containing medium for 2 ml of normal growth medium (not yet containing puromycin).


Around 3-4 days post-transfection, harvest the cells of one well and wash them once in normal growth medium supplemented with 10 µg/ml puromycin. Resuspend the cells in 5 ml of selective medium. Transfer the cells into a 15 ml flask (batch selection).

After 1-2 weeks, cells should start growing. If so, expand them and test for expression of your transgene.