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

Because human casein does not precipitate from milk at its isoelectric poin t as does bovine casein, there is no easy method of quantitation. Casein re presents only approximately 30% of the protein fraction in human milk, and the complex methods necessary for isolation cannot be used easily with smal l samples in a survey of a large number of mothers. Two-dimensional electro phoresis coupled with computer imaging has the potential to compare and qua ntitate proteins expeditiously using a small sample size. Iso-Dalt, a denat uring methodology, separates the casein micelle into its component parts, b eta-casein, kappa-casein, parakappa-casein and casomorphins. Identification of these spots was made by immunoassay of a Western blot with monoclonal a nti-human casein. Two spots at 24 kDa and 26 kDa, thought to be phosphoryla ted isomers of beta casein, were selected for quantitation. Milk samples fr om 20 mothers, 8 weeks post partum, were run on two-dimensional (2-D) gels; a slide was taken of each silver-stained gel with a Kodak control strip; t he 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 an d integrated density of the spots were measured. The Kodak control scale pr ovided 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 c asein (Sigma). Measurements will be used in a statistical program, Statview (Abacus), in an attempt to correlate information from a questionaire with casein concentration.