| Popis: |
A sensitive and specific immunoassay for mouse syndecan-4 is described. The assay is a non-competitive direct sandwich enzyme-linked immunosorbent assay based on the production of an affinity-purified polyclonal antibody directed against syndecan-4 extracellular domain, used both as a trapping and detecting antibody. The standard curve is constructed with recombinant 6-His-tagged syndecan-4 extracellular domain. The assay allowed quantification of syndecan-4 core protein in the partially purified proteoglycan fraction from adipocyte 3T3-F442A cells, as well as in the cellular whole and clarified lysates. Removal of glycosaminoglycan chains on syndecan-4 core protein is required for maximal epitope exposure. The standard curve ranged between 7 and 70 fmol per well. For the 14 standard curves run on different days, the absorbance at 490 nm for 35.5 fmol of recombinant syndecan-4 was 0.610 ± 0.110 (n = 14) with a corresponding blank absorbance of 0.089 ± 0.030. At low (5.7 fmol) and high (42.8 fmol) levels of whole cell syndecan-4, the intra-assay and inter-assay coefficients of variation were 4.9 and 15.3 percent, and 3.3 and 9.7 percent, respectively. A survey of the syndecan-4 expression in various mouse tissues shows that syndecan-4 is highly expressed in the kidney, brain, testes and liver, but can also be measured in several different adipose tissue sites. —Rioux, R., R. Y. Landry, and A. Bensadoun. Sandwich immunoassay for the measurement of murine syndecan-4. J. Lipid Res. 2002. 43: 167–173. |
