Help:how to get better resolution using FPLC? - (Jul/24/2008 )

I am using HiLoad 16/60 superdex 200 prep grade column from GE Healthcare to run FPLC for protein standards seperation. I tried some times and found that two peaks always merged. The standards should be ok becos somebody used these proteins to get perfect data some years ago. If I want to get sharp peaks and wide retention time between peaks, what can I improve my FPLC? My buffer is TBS/EDTA with PH 7.5. Could anyone give me some suggestion on it? thanks!

[quote name='3203290' date='Jul 24 2008, 12:11 AM' post='144956']
I am using HiLoad 16/60 superdex 200 prep grade column from GE Healthcare to run FPLC for protein standards seperation. I tried some times and found that two peaks always merged. The standards should be ok becos somebody used these proteins to get perfect data some years ago. If I want to get sharp peaks and wide retention time between peaks, what can I improve my FPLC? My buffer is TBS/EDTA with PH 7.5. Could anyone give me some suggestion on it? thanks!
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what are the MWs of the two proteins? usually by reducing the loading volume and not to run too fast will help improving the resolution. but if the two proteins are too close, there is a limit of this methodology can do for you.

Have you tried PBS instead of TBS? Also, try increasing the salt further to 200 mM or even 500 mM.

you could try altering pH that u used beside try with other type of column.

thanks for your advice! I adjusted flow rate and got better resoluton. I have another problem with my FPLC. I am using Ferritin (440kd), IgG (158kd), Albumin (67kd), Ovalbumin (43kd) and Myoglobin (17kd) as my standards. I always got an unknown small peak between Ferritin and IgG. I tested individual for all standards and found that this peak appeared nearly at the same retention time. I ran blank (only running buffer) but I didn't get this peak. That means my column is clean. My colleagues think they are aggregates of my proteins. The column datasheet also suggests a aggregate peak before Ferritin. However, in my FPLC they came out slower than Ferritin and had the same retention time no matther what standard I used. I can't figure out how to remove it? Do you have any idea on it? Thanks in advance!