I have managed to isolate LDL using dialysis & sequential ultracentifugation and have used the Lowry assay to quantify the amount of protein in the sample. Is it possible to concentrate LDL without causing aggregation or losing too much sample in the process?
We do, it's rather easy using the "concentrating solution" and dialysis cassettes (Slide a lyzer, 10K MWCO) of Thermo fisher scientific. After putting the lipoproteins in the concentrating solution you can measure the protein concentration again using a BCA kit. If you want a more detailed protocol, just let me know.
Hi,
you can use VivaSpin Columns from GE Healthcare. Its fast and simple.
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/de/GELifeSciences/products/AlternativeProductStructure_17079/28932218
Hi Michael,
I wasn't sure if the VivaSpin columns would cause the protein to aggregate?
If you want concentrated LDL , after the first ultacentrifugation step, the QC is a second step by an electrophoresis on agarose gel. You must obtain only one LDL acute peak after stainnig it with Red or Black oil. The third step is a second separation , as Sven says , by a new ultracentrifugation at 16 Centigrade degrees with a Density 1.15 g/mL PBS solution , to obtain a floating LDL fraction on the top of the tube. To be sure that your LDL fraction is concentrated, you can run a measurement of protein level by a standard method or an ApoB lipoprotein immunoelectrophoresis with ApoB ac., as compared with the first LDL fraction If you want to separate small dense LDL see the present attachment
A new approach to the quantitative measurement of dense LDL subfractions.
Berg G, Muzzio ML, Wikinski R, Schreier L.
Nutr Metab Cardiovasc Dis. 2004 Apr;14(2):73-80.
PMID:15242239[PubMed - indexed for MEDLINE]
I agree with Sven and Regina. For me, the best method is a second UC in a homogeneous medium density (KBr) of 1.065 g / ml, placing your LDL solution in the bottom of the tube. With this approach it might be a slight contamination of Lp (a) if you have in your sample is low density (unlikely), but avoid contamination with Lp (a) denser (than usual)
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