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The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA) and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)n oligonucleotides using surface plasmon resonance spectroscopy. A single NC protein is able to bind to more than one immobilized oligonucleotide. However, if oligonucleotides are immobilized at low densities (~10RUs), then NC no longer displays this behavior. As NC is proposed to be the nucleic acid binding domain of Gag, we would expect Gag to show the same behavior. To investigate the stoichiometry and cooperativity of Gag binding to oligonucleotides, we must ensure that Gag is unable to bind to two oligonucleotides at once. In addition, we need to know accurately how much oligonucleotide is immobilized so we can calculate the stoichiometry of Gag binding. We developed a method where we can determine precisely the amount of oligonucleotide immobilized at very low-density surfaces (0.1–2 RUs). In this approach, we used electrospray ionization Fourier transform mass spectrometry to determine that two and four molecules of NC bind to d(TG)5 and d(TG)10. We were then able to use NC injections to calibrate the immobilized oligonucleotide and determine an accurate measurement of the surface density. Using this approach, we have measured the binding of Gag to d(TG)n. Gag binds to a 5-mer and a 20-mer with a stoichiometry greater than 1 and 4, respectively. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules bind to the Gag already bound to the oligonucleotide, forming oligo-Gag-Gag complexes.