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Denatured, reduced Western blotting (100 ng or less) as a standard only. This material is not recommended for ELISA or ANY other application. Other applications have not been tested. Please follow the Western blot resuspension and handling notes in the instructions included below for the best results:
On reduced SDS-PAGE gels, beta caseins migrate at approximately 24-26 kDa. Note that different gel conditions can cause beta caseins to migrate slower than normal, sometimes as high as 33-35 kDa. The recommended gel is 4-20% Bis-Tris Gel with MOPS as a running buffer.
For testing raw milk samples, stock milk must be first diluted. Dilute raw milk in 0.1 M NaOH at 1:50. Then mix 10 µl of diluted milk with 10 µl 2X SDS-PAGE (with DTT) sample buffer and boil for 5 minutes, spin, and load 10 µl/lane. For purified A1 or A2 protein, mix 10 µl of prewarmed and mixed protein from the unopened vial with 10 µl 2X SDS-PAGE (with DTT) sample buffer, boil for 5 minutes, spin, and load 8 µl/lane.
Note on handling: Purified beta caseins can and will precipitate out of the solution when cold or below pH 8. Before use in a Western blot, the unopened vials should be warmed to 37°C, given a quick spin, and gently mixed with a pipette tip or vortex to ensure the precipitated protein has been redissolved.
Purified Caseins are very sensitive to acidic conditions. At pH under pH 8, they will easily precipitate or aggregate. The user is advised to keep this in mind when working with these proteins. Purified caseins are insoluble in neutral water. They solubilize in alkali salts such as sodium acetate or sodium oxalate at pH readings over pH 8. Care in handling and storing these purified proteins is advised.
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