Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting.
The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.
Bicsak
T A
TA
Whittier Institute for Diabetes and Endocrinology, Department of Molecular Endocrinology, La Jolla, California 92037.
Nakatani
A
A
Shimonaka
M
M
Malkowski
M
M
Ling
N
N
D000818:Animals; D002352:Carrier Proteins / Q000097:blood / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015151:Immunoblotting; D018969:Insulin-Like Growth Factor Binding Proteins; D008024:Ligands; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism*; D013002:Somatomedins / Q000378:metabolism*
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D008024:Ligands; D013002:Somatomedins; D012967:Sodium Dodecyl Sulfate