Purification of porcine beta-casein, N-terminal sequence, quantification in mastitic milk.
The objectives of the study were to purify porcine beta-casein from sow's milk, to determine N-terminal amino acid sequence, to develop specific antisera against porcine beta-casein, and to use that antisera to evaluate milk samples from a mastitis study. Milk was collected by hand milking a Yorkshire by Duroc crossbred sow following oxytocin administration on d 27 of lactation. A casein-enriched fraction was then prepared by iso-electric precipitation. Porcine beta-casein was then purified by liquid chromatography on a Mono Q anion-exchange column, and checked for purity with SDS-PAGE. An apparent molecular weight of 29,000 Da was estimated from SDS-PAGE. N-Terminal amino acid sequence was determined by Edman degradation to be RAKEELNASGETVE. Rabbits (n = 2) were immunized with beta-casein mixed with Freund's complete (primary) or incomplete (boosters) adjuvant at 4-wk intervals. Antiserum collected from one rabbit 112 d after primary immunization detected 30 to 100 ng beta-casein by Western blot procedure when used at a dilution of 1:2 x 10(6). The antiserum was specific for porcine beta-casein, but showed some cross-reactivity with equine casein. It was determined by Western blot procedure that mammary inflammation induced by lipopolysaccharide infusion resulted in a 41% decrease in the beta-casein concentration of sow milk.
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002364:Caseins / Q000737:chemistry / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000662:veterinary; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D005620:Freund's Adjuvant; D008413:Mastitis / Q000382:microbiology / Q000503:physiopathology / Q000662:veterinary*; D008892:Milk / Q000737:chemistry / Q000382:microbiology*; D008970:Molecular Weight; D011817:Rabbits; D013552:Swine / Q000502:physiology; D013553:Swine Diseases / Q000382:microbiology* / Q000503:physiopathology
D002364:Caseins; D005620:Freund's Adjuvant