size exclusion.. how to reduce tailing. for proteins.. - sillica matrix tsk columns.. (Aug/06/2008 )
hi all.. i m working on sec with proteins..
i've tried acetate buffer.. phosphate buffer.. both with and without salt.. with and without 10% ethanol.. my protein gives a lot of tailing.. this is on a sillica matrix tsk columns.. any suggestions??
-alice!-
According to "Gel Filtration. Principles and Methods" from Amersham Biosciences:
QUOTE
Tailing peaks
Asymmetric peaks: sample application uneven. Check top of column if possible. Ensure medium is evenly packed and that sample is applied without disturbing the packed bed. Tailing peaks can also be due to underpacking of column (packed at too low pressure or flow rate).
Asymmetric peaks: sample application uneven. Check top of column if possible. Ensure medium is evenly packed and that sample is applied without disturbing the packed bed. Tailing peaks can also be due to underpacking of column (packed at too low pressure or flow rate).
-K.B.-
QUOTE (alice! @ Aug 7 2008, 02:51 AM)
hi all.. i m working on sec with proteins..
i've tried acetate buffer.. phosphate buffer.. both with and without salt.. with and without 10% ethanol.. my protein gives a lot of tailing.. this is on a sillica matrix tsk columns.. any suggestions??
i've tried acetate buffer.. phosphate buffer.. both with and without salt.. with and without 10% ethanol.. my protein gives a lot of tailing.. this is on a sillica matrix tsk columns.. any suggestions??
Have you tried running size standards such as BSA, chymotrypsin proteins down the same column under the same buffer conditions (GE life sciences sell low or high MW calibration kits which contains different proteins for the purpose of calibration a SEC column). These normally give rise to roughly symmetrical peaks (you should have 1mM reducing agent such as Dithiothreitol present in your buffer though as these proteins have exposed cysteines). If the peaks are not symmetrical then the problem lies in the resin/column packing.
However long tailing could be a sign that your protein self associates with a relatively weak interaction so as the protein passes down the column and becomes diluted by axial dispersion the large protein species dissociate into smaller species.
Try running different concentrations of your protein down the column keeping the injection volume, flow rate, buffer... constant. If the protein has multiple oligomeric states you will see the elution peak shift to the right as the loaded protein concentration is reduced.
for an example of this see
Smyth et al 2000 Molecular Microbiology Volume 36 Issue 4, Pages 962 - 972
-leonardp-