|Abstract:||High-density lipoproteins (HDL) are complex particles composed
of lipids and apoliproteins. The major function of HDL is to
transport cholesterol from peripheral tissues to the liver. HDL
apolipoproteins include apoAI, a major structural apolipoprotein;
apoCIII, a protein that inhibits binding of apoE and apoB-100 to
hepatic receptors; and apoE, a ligand for the low-density lipoprotein
(LDL) receptor, apoE receptor, and LDL receptor related protein.
Immunoaffinity column chromatography methods are frequently
used to isolate HDL subspecies. Therefore, it is important
to understand how elution buffers may be affecting protein
detection and potentially denaturing proteins. In order to optimize
immunoaffinity column chromatography elution conditions,
we tested the effects of two elution buffers, acetic acid and sodium
thiocyanate (NaSCN), on apoA1, apoCIII, and apoE. We tested
the elution buffers for: number of elutions required to elute
protein, protein recovery, and reduction of protein detection after
treatment with elution buffer. Plasma samples containing known
concentrations of apoliproteins listed were used. This allowed us
to compare expected concentrations and measured concentrations
after treatment. The sandwich enzyme-linked immunosorbent
assay (ELISA) method was used to quantify protein concentrations.
The results of these experiments will allow us to maximize protein recovery and account for loss of protein detection due to treatment with elution buffers.