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Simberg and co-workers introduced a model for the protein corona which includes "primary binders" that interact with the NPs surface at first followed by "secondary binders" that binds to the primary binders by way of protein protein interactions [39].
DNase I footprinting analysis of DevRC with the tgs1-Rv3131 interegion revealedevealed that it binds to the primary site, but fails to cooperatively bind to the adjacent site, unlike full-length DevR which protected both the sites (Fig. 2).
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Briefly, depletion of CD4-negative T cells was achieved by incubation of peripheral blood mononuclear cells with a cocktail of monoclonal antibodies directed against non-T cells and against CD4-negative T cells, followed by binding of a secondary microbead-coupled antibody to bind to the primary antibodies.
Furthermore, we demonstrate that HIV and SIV Nef bind to the primary viral receptor CD4 with comparable efficiency.
The secondary antibody Goat Anti-Mouse IgG AP conjugate bound to the primary antibody (IgG) and the signal was enhanced.
After incubation and washing, strong magnets were used to remove all beads that bound to the primary or secondary antibody.
A secondary antibody coupled to horseradish peroxidase (1 10,000; Amersham Biosciences) was used to bind to the primary antibody and be detected using enhanced chemiluminescence (NEN Life Science Products).
The embryos were also incubated with commercially available secondary antibodies that bind to the primary antibodies, and that are cross-linked to fluorescent dyes (Alexa488, Alexa555 and Alexa647).
Species-specific secondary antibodies, called PLA probes, each with a unique short DNA strand attached to it, bind to the primary antibodies.
This is primarily because conventional aptamer discovery methods (i.e., SELEX) have a strong tendency to only yield aptamers that preferentially bind to the primary, dominant epitope at the expense of other aptamers that bind to secondary, nondominant epitopes.
Thus, a collagen-like peptide with only two GVMGFO motifs may be unable to activate DDR on cells, even though it would still bind to the primary site at the top of the DS domain.
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