Rapid screening method for polymorphism of group A apolipoproteins.
Polymorphism of apolipoproteins AI and AII (apo AI and apo AII) can be easily investigated in plasma by a simple method involving a 30-min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P-40 followed by subsequent isoelectric focusing (IEF). The sample (2 microL) was applied to an ultrathin flat acrylamide gel of pH range 4-6, and focused using a Bio-Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R-250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins after IEF, the gel was immunofixed directly with anti-apo AI, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo AI and apo AII and an anti-immunoglobulin-alkaline phosphatase conjugate. High-density lipoprotein (HDL) was used as a standard for Apo AI variants. Employing these techniques, human plasma apo AI was resolved into one major band (apo AI0, pI 5.54), and four minor bands identified as apo AI+2 (pI 5.75), apo AI+1 (pI 5.66), apo AI-1 (pI 5.45), and apo AI-2 (pI 5.34). Apo AII was resolved into one major isoprotein designated as apo AII0 (pI 4.87), and two minor isoforms apo AII+1 and apo AII-1 which focused at pIs of 5.18 and 4.58, respectively. The results showed that these methods can be used to identify apo AI and AII isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, fixation (chemical or immunofixation), and staining, can be accomplished in 5 h compared to 2 days using previously reported technique.(ABSTRACT TRUNCATED AT 250 WORDS)
Harake
B
B
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Caines
P S
PS
Thibert
R J
RJ
D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016633:Apolipoprotein A-II / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D011110:Polymorphism, Genetic
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II