Two-dimensional electrophoresis and computer imaging: quantitation of human milk casein

Because human casein does not precipitate from milk at its isoelectric point as does bovine casein, there is no easy method of quantitation. Casein represents only approximately 30% of the protein fraction in human milk, and the complex methods necessary for isolation cannot be used easily with small samples in a survey of a large number of mothers. Two-dimensional electrophoresis coupled with computer imaging has the potential to compare and quantitate proteins expeditiously using a small sample size. IsoDalt, a denaturing methodology, separates the casein micelle into its component parts, beta-casein, kappa-casein, parakappa-casein and casomorphins. Identification of these spots was made by immunoassay of a Western blot with monoclonal anti-human casein. Two spots at 24 kDa and 26 kDa, thought to be phosphorylated isomers of beta casein, were selected for quantitation. Milk samples from 20 mothers, 8 weeks post partum, were run on two-dimensional (2-D) gels; a slide was taken of each silverstained gel with a Kodak control strip; the slide was scanned into powerMac Photoshop 3 with a Polaroid-Sprintscan; spots were isolated using "threshold", "mask" with IPTK (Imaging Processing Tool Kit, Reindeer Games) a Photoshop plug-in, and transferred to the NIH-Image program. Using an NIH-Image gel macro (Thomas Seebacher), the area and integrated density of the spots were measured. The Kodak control scale provided calibration and conversion to OD units. Visual scanning of the gels and computer units indicated a wide range of concentrations. To understand the range in units of weight, a standard was generated using bovine alpha casein (Sigma). Measurements will be used in a statistical program, Statview (Abacus), in an attempt to correlate information from a questionaire with casein concentration.