(Autoradiogram/Phosphorimage) Fig. 1. Analytical ACE schematic. Top panel: ACE gels poured using a casting stand and Teflon combs and strips as shown in Fig. 5 are used to create nine parallel rectangular wells, which are filled with protein-agarose mixtures, each at a different protein concentration. Radiolabeled GAG or PG is loaded into the slot above the protein-containing wells (shown as a dark line), and after electrophoresis of the GAG or PG through the protein-containing lanes, its migration as a function of protein concentration is visualized by autoradiography or phosphorimaging (shown here as a pattern of peaks and valleys). The degree of GAG/PG retardation at the various protein concentrations is used to calculate the apparent Kd of GAG- or PG-protein binding (see text for details). Artwork by Shawn M. Sweeney.
Fig. 2. (opposite page) ACE analysis can reveal the affinity of interactions between PGs or GAGs and various proteins. For these experiments, syndecan-1 was electrophoresed through types I-VI collagens in ACE gels. (A) Images of PG migration patterns were obtained using a phosphorimager. The electrophoretograms indicate that some collagens bind strongly to syndecan-1 (e.g., type V), and others bind weakly (e.g., type II). Protein concentrations in nM are shown beneath gels. (B) Calculation of affinities of syndecan-1 for various human collagens. From each electrophoretogram in panel (A), retardation coefficients (R) for syndecan-1 were determined (see text) and are plotted against protein concentration. Smooth curves represent nonlinear least-squares fits to the equation R = R^ (1 + (Kd/[protein])2). Data are adapted from (5).
Fig. 3. ACE analysis can reveal selectivity in PG- or GAG-protein interactions. Example of ACE analysis of the interactions between a basic peptide and 35S-sulfate metabolically labeled PGs/GAGs secreted by endothelial cells in vitro. ACE gel image was obtained using a phosphorimager. At least two populations of PG/GAG, seen as two bands of radiolabeled material migrating with different mobilities at low protein concentrations (< 50 nM), indicates heterogeneity in size and/or charge density within the PG/GAG mixture. Potential heterogeneity in PG/GAG-peptide interactions is also obvious at a peptide concentration of 250 nM, in which a fractionation of the PG species through the peptide-containing lane is evident as a broad smear throughout the lane, and as a sharp band that migrates approximately halfway down the lane. Thus, components of the PG/GAG sample are binding strongly to the peptide (i.e., are retained closer to the top of the peptide-containing lane), and others are binding more weakly to the peptide (i.e., are not significantly retained and migrate further within the peptide-contain-ing lane). In such cases preparative ACE can be used to recover differentially binding PG/GAG populations for further characterization. Data are adapted from (11).
1.1. Do I Have Enough Protein?
There is no way of knowing a priori how much of a protein sample one needs for an ACE gel, since it depends on a yet to be determined value, i.e., the affinity the protein will exhibit for GAGs or PGs. However, some general idea about the amounts of protein required can be derived from the following example. For type I collagen, a protein of Mr ^ 300,000 Da that exhibits a heparin-binding Kd in the range of 100-200 nM, one needs 150 ^g of protein per ACE gel (using the ACE gel dimensions specified under Subheading 2.). This amount allows for the creation of nine protein-agarose samples of 250-^L each, at concentrations of 1000, 500, 250, 100, 50, 25, 10, 5, and 1 nM. Since ACE gels should be repeated at least three times to derive a reasonable estimate of the Kd, then one would need a minimum of 450 ^g of type I collagen for three experiments. Other proteins such as growth factors are much smaller than collagens, and often exhibit much higher affinities for GAGs and PGs, and thus can require considerably less protein for three experiments, i.e., on average < 50 ^g total, or in some cases even much less—e.g., for basic fibroblast growth factor, < 1 ^g is required.
1.2. Will the Protein Remain Native?
The native state of the protein, its solubility, and propensity to aggregate as a function of its concentration or solvent are key considerations, and must be determined for each protein used in ACE. For example, in the case of laminin, which tends to aggregate in solution, ethylendiamminetetraacetic acid (EDTA) can be supplemented to the protein samples and the ACE buffers to inhibit aggregation (3). In the case of the collagens,
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