We report in this article a systematic study on the effect of overall grafting density (sigma(overall)) on microphase separation of mixed poly(tert-butyl acrylate) (PtBA)/polystyrene (PS) brushes synthesized from asymmetric difunctional initiator (Y-initiator)-functionalized silica particles. The initiator particles were made by the immobilization of a triethoxysilane-terminated Y-initiator on the surface of 173 nm silica particles via an ammonia-catalyzed hydrolysis/condensation process in ethanol. Mixed PtBA/PS brushes were then grown from initiator particles by surface-initiated atom transfer radical polymerization of tBA at 75 degrees C and nitroxide-mediated radical polymerization of styrene at 120 degrees C. By changing the mass ratio of Y-initiator to silica particles in the initiator immobilization step, we found that the overall grafting density of mixed brushes can be systematically tuned while the individual grafting densities of two polymers remained comparable. A series of mixed PtBA/PS brushes with molecular weights of similar to 23 kDa for both PtBA and PS and sigma(overall) from 1.06 to 0.122 chains/nm(2) were prepared. Their microphase separation behavior was studied by transmission electron microscopy after the samples were drop-cast from chloroform onto carbon-coated TEM grids, annealed with CHCl3 vapor, and stained with RuO4. CHCl3 is a good solvent for both grafted polymers. When the sigma(overall) was 0.34 chains/nm(2) and above, the mixed PtBA/PS brushes underwent lateral microphase separation, forming "rippled" nanostructures. The ripple wavelength (D) increased with decreasing sigma(overall); the normalized D scaled with sigma(-0.47)(overall) in the sigma(overall) range of 1.06-0.54 chains/nm(2). With further decreasing the grafting density, the phase separation became weaker, and no microphase separation was observed in the sample with sigma(overall) of 0.122 chain/nm(2).