A method for detecting different β-casein variants in bovine milk
A casein and variant technology, applied in the field of animal breeding and food testing, can solve problems such as false positive results, and achieve the effects of simple operation, reasonable design, and huge economic and social benefits
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Embodiment 1
[0072] Example 1: Construction of a method for detecting different β-casein variants
[0073] 1. Protein extraction and quality control of milk or milk powder samples.
[0074] 8M urea was used to fully dissolve the protein in the sample, the protein and oil in the sample were separated by centrifugation, and the obtained protein sample was subjected to dithiothreitol (DTT) reduction and iodoacetamide (IAM) alkylation protection. Coomassie brilliant blue staining (bradford method) was then used to quantify the protein, and sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the protein band was clear without degradation.
[0075] 2. Carry out enzymatic digestion to the above-mentioned extracted protein;
[0076] Get appropriate amount of above-mentioned sample, utilize the method for ultracentrifugation, the buffer solution in the sample is changed into 25mM ammonium bicarbonate (NH 4 HCO 3 ), in order to facilitate the subsequ...
Embodiment 2
[0081] Embodiment 2: The accuracy and repeatability detection of the method.
[0082] The milk cow whose β-casein genotype has been determined through direct DNA sequencing was used as the detection object, and the method of Example 1 was used to identify different variants of β-casein in milk, and the accuracy rate was 100%. It shows that the detection method is accurate and effective.
[0083] Taking milk samples with known β-casein variants as the detection object, this detection method was used for repeated tests, and the results of multiple repeated tests were consistent, indicating that this method has good stability.
Embodiment 3
[0084] Example 3: Detection of actual samples.
[0085] The method of Example 1 of the present invention was used to detect two raw milk samples.
[0086] 1. Protein extraction and quality control;
[0087] (1) Take 50 μl of raw milk sample and add 500 μl of lysate (8M urea, 30mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes)). Lyse on ice for 10 min. Centrifuge at 4°C, 20000g, 30min, take the supernatant, avoid absorbing oil.
[0088] (2) Add dithiothreitol (DTT) to a final concentration of 10 mM. 56°C water bath for 1h. After taking it out, add iodoacetamide (IAM) quickly to a final concentration of 55mM, and let it stand in a dark room for 1h.
[0089] (3) Proteins were quantified using the Coomassie blue staining method (Bradford method).
[0090] (4) Carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to the protein after quantification ( figure 2 ).
[0091] 2. Carry out proteolytic digestion of the above-mentioned extracted prote...
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