Figure 7.

SDS-PAGE and subsequent immunoblot analysis of lipoproteins. Lipoproteins (200 μg) in a total volume of 200 μl were incubated with or without calcium (10 μM) in the absence or presence of variable concentrations of glucosamine hydrochloride for 4 hours at 30°C. The reaction was stopped by the addition of EDTA (ethylenediaminetetraacetic acid) with butylated hydroxytoluene, and aliquots were stored at -70°C. Thawed samples were derivatized with DNP, and 40 μl of sample was loaded on SDS-PAGE and transblotted onto nitrocellulose membranes. Subsequently, the membrane was incubated with anti-DNP antibodies for 1 hour and processed. DNP, dinitrophenyl; Glu, glucosamine hydrochloride; LDL, low-density lipoprotein; M.W., molecular weight; Vit E, vitamin E. Arrow indicates diffused DNP reactivity to high molecular weight lipoproteins in Lane 3.