FPLC fractionation of lipoproteins (to identify the amounts of VLDL, HDL in seru - FPLC fractionation of serum (Jun/09/2007 )

Hi everyone,
I'm Annabel, a fourth year Ph.D, focusing on hepatic lipogenic regulation. Right now I need to apply liquid chromatography fractionation of lipoproteins, using mice plasma or serum. Unfortunately, I know nothing about the FPLC system, does any one here know the precise procedure of doing that? I have read through several papers, but they don’t share the same conditions. I want to know how much sample should be loaded, the constant flow rate and the time to get fractions. Our cloumn is Tricorn high-performance Superose S-6 10/300GL.
Thank you!
Annabel

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Dear annabel!

The volume of your column is 24 ml , for proper GF procedure you should use sample volume = 1% column volume/ So you should load max 250ul, but it will be better to apply for first start 50 to 100 ul to estimate resolution of your protein mixture separation

Flow rate - it will be better 0,2-0,3 ml\min. Time with this flow rate easy to calculate = 24 [mL] / 0,2 [mL/min] = 120 min (It is max possible time you should wait for your last peak elution) (near 1.5-2 hours for all procedure and 1 chromatography profile).
Filtrate your sample through 0,2um or centrifuge your sample 10min at max speed (eppendorf tube centrifuge will give you near 16000g).
Control pressure on your HPLC system, don.t exceed 350psi.
Fill all your tubing before column with eluation buffer and don't forget to filtrate your buffer on 0.45um and degass, if your HPLC system haven't build in degassator.

GooD LUCK!

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