Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey-based -lactalbumin (-La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure -La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. -La and, additionally -Lg, were purified at the same batches with the purities in the range of 95%-99%. After enzymatic hydrolysis, WPI-based -La produced chain-like and long nanotubules with approximate to 20 nm width while WPC-based -La produced thinner, miscellaneous, and fibril-like nanostructures by self-assembly. Raman and FT-IR spectroscopies revealed that -La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy-based -La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1301-1310, 2014