Anindito,
First off, I have never tried this, but here are a few initial thoughts on how I might approach this. But first a question, how tight is the dimer form? if you mixed dimers with tags and dimers without tags, would you get any exchange? If so, you might be able to use this to your advantage. But for now, i'll ignore this. Now for how i was thinking of possibly accomplishing the goal you want... 1- use two vectors or a DUET vector to express two versions of your protein, (1) with N-term cleavable (TEV site?) HIS tag and your fusion protein, and (2) with N-term cleavable (TEV site?) GST tag only. 2- express your protein as normal and then purify by (1) running the GSH column to capture all GST tagged proteins, followed by a (2) Ni-NTA column to capture those with HIS tags. If all works as planned, you would end up with one monomer having only HIS-fusion protein tags and one having GST tag. You could use SDS-PAGE/Western blot analysis to verify this, since the monomers will run at different sizes (you might have to modify your gel system to get the best separation....i recommend 5-7% gels for this size in MES) AND should be reactive to antiHIS and antiGST antibodies in needed. Further, you could get an idea for the ratios of each by comparing the overall intensity of the bands under normal staining methods. 3- to remove the HIS and GST tags, just treat with TEV protease to yield your dimer with only one of the monomers containing your fusion protein. I am sure i likely overlooked something in my haste, but you get the idea, basically a two step purification with two tags. Again, I haven't tried this but seems like it should work to me. hope this helps, good luck! Cheers, Nick -------------------------------- [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ ________________________________________ From: Anindito Sen [andysen.to...@gmail.com] Sent: Monday, August 26, 2013 11:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex Dear All, I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer protein (size is ~100kDa). To be more precise - The tag need to get itself attached only on one of the dimer molecules. My expertise are not in Cell biology and therefore any suggestion in this regard will be of great help. Thanks & Best Wishes Dr. Anindito Sen (Ph.D) Structural Biology Graduate School of Medicine University of Tokyo 7-3-1 Hongo. Bunkyo-ku. Tokyo 113-0033. Japan Tel & Fax: +81-3-5841-3339
