Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay.
It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less.
Nishibu
Takahiro
T
Diagnostic Laboratory Osaka, Wako Pure Chemical Industries, Ltd., 1-6, Takadacho, Amagasaki, Hyogo 661-0963, Japan.
Hirayasu
Kazunari
K
Tanaka
Takumi
T
Takeda
Yasuhiro
Y
Kobayashi
Yoshiteru
Y
D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D005374:Filtration; D011506:Proteins / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate; D012996:Solutions
D011506:Proteins; D012996:Solutions; D012967:Sodium Dodecyl Sulfate