I am working with two different proteins; which were proven to interact with each other using in vitro pull down, co-ip (both with recombinant proteins and from in-vivo system) studies. preliminary elisa tests showed the affinity to be roughly around 60uM (Kd). in an effort to study the interacting interface (still not known between the two proteins) I tried to form the complex of the two proteins on the GFC- gel filtration column (superdex/ superose) but after trying all the different approaches i could not purify the complex in GFC(no shift in the elution profile). it seems like the interaction between the two proteins is either weak (not enough to sustain GFC runs) or very transient or dyanmic. is there any other way to get the complex (have tried GraFix technique for complex formation and SPR for checking the thermodynamics of interactions but not much success) or ways to prove that the interaction transient or dynamic etc?
Equiliberate the column with mobile phase containing one of the proteins which is relatively cheaper. Load the column with second protein and resolve the complex using mobile phase containing first protein. Collect eluted fractions and some of the fractions will contain protein-protein complex. This must be the first to elute than each of the individual proteins being of highest Mr.
Hi there,
You should try ITC provided you dispose of enough pure material.
Hello, Size exclusion chromatography separates proteins by their hydrodynamic radius, a property determined by both the size and shape of the molecule. Proteins do not bind to a stationary phase are separated by the speed at which they navigate through an inert stationary phase.
For more info use link below
Good luck!
https://www.labome.com/method/Protein-Purification.html
Shamsher S. Kanwar Hi, I am not sure if I will be able to spend so much protein (to be used as a mobile phase for my runs), but thank you for your reply.
Dominique Liger I will see if i can get my hands to nearby facility that can offer me ITC services. Thank you.
Sebastian Schmitt
. Hi I have verified the interaction of my recombinantly purified proteins using IP and other techniques. The buffer conditions are same in all the cases (as well as bacterial expression systems) and even my FPLC runs. The protein yield is optimised to give good and high amount with pure fractions for both the proteins and Another thing that I am trying currently is to stabilize the interaction by using some known interacting partners of both the proteins. I am also considering to cross linking the two proteins. Thank you for the replyKaren A. Darbinyan thank you for your reply.
Sourav Roy
. hi , i considered the kD value and used almost 0.3 to 0.5mM of my protein for s200 (120mL columns), but somehow it did not help much. May be i will have to prove that the interaction is quite transient and dynamic enough to be stabilized on the columns :(
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