An improved method for determining high molecular weights by protein immunoblotting: application to apolipoprotein(a) phenotyping.
The determination of very high molecular weights (greater than 500 kd) by protein immunoblotting is limited by a lack of commercially available molecular weight standards in that range. We have therefore investigated the use of the human haptoglobin 2-2 phenotype polymeric series as a high molecular weight standard curve. Using pre-electrophoresed 3-12% SDS polyacrylamide gradient gels, non-dissociated haptoglobin 2-2 serum was separated, transferred and probed with anti-human haptoglobin. We were able to visualize up to 13 bands, with a theoretical molecular weight range of 171.9-859.5 kd, in non-dissociated haptoglobin 2-2 serum. Molecular weights up to 584 kd were confirmed by comparison with available standards. The method was then applied to the determination of apolipoprotein(a) [apo(a)] phenotype molecular weights. Precision of the method was excellent, with intra-run CVs of less than 2.9% and inter-run CVs of less than 3.2% for apo(a) molecular weight. This method of molecular weight calibration is applicable to the characterization of any high molecular weight protein that can be successfully electrophoresed, transferred and visualized.
Craig
W Y
WY
Foundation for Blood Research, Scarborough, Maine 04070-0190.
Forster
N R
NR
Ledue
T B
TB
Johnson
A M
AM
Neveux
L M
LM
Ritchie
R F
RF
D001054:Apolipoproteins A / Q000032:analysis* / Q000592:standards; D015415:Biomarkers; D004591:Electrophoresis, Polyacrylamide Gel; D006242:Haptoglobins / Q000032:analysis / Q000592:standards; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D010641:Phenotype; D012015:Reference Standards
D001054:Apolipoproteins A; D015415:Biomarkers; D006242:Haptoglobins