I am using HiLoad Superdex 26/600 SEC for polishing. I need to run 50mM Tris pH 7.5, 1mM EDTA, 100mM Tris and 10% Glycerol to simultaneously polish and purify my protein.
Can glycerol be used on such column?
I am working at temperature of 10 degree Celsius.
I would be greatly helped with suggestions.
Generally, it can. It is not uncommon to have 10 % glycerol in SEC buffer (including Superdex 75 columns). However, if using glycerol you need to set the flow rate low to not exceed pressure limit of the column. 10 deg C also is not a problem as long as you keep the pressure in the limits.
Yes, you can use 10% glycerol, but normally 10% is the maximum of glycerol that you can use. You will have to low the flow rate or put an automatic flow rate control with the pressure.
Agree with Wojciech and Jaime, if the flow rate is too fast you will compress the column bed/media and then you will have to repack the column. So when you are equilibrating the column with the 10% glycerol buffer keep an eye on the filter at the top of the column for any early signs of compression (will see a gab appear below the filter and above the bed). If you see the bed start to compress stop the flow and undo a fitting between the pump and column to release the pressure and then the column bed may return to the "approximate" original position in 1-2 hrs to O/N.
When you load your protein it will have a greater back pressure effect than buffer alone, so you may want to load it at a flow rate that is less that what works for the buffer alone (50% less), once the protein is in the media you should be able to return to the normal flow rate.
So set the back pressure on the pumps to a little less than what is acceptable for the column to protect it. The colder the buffer/column the greater the back pressure.
Yes, you can use glycerol for your Superdex column, but you should not use more than 10% of it.
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