"A2 Milk" Authentication by Isoelectric Focusing and PCR Techniques (CAT#: STEM-ET-0349-ZJF)

Name: "A2 Milk" Authentication by Isoelectric Focusing and PCR Techniques
SKU: STEM-ET-0349-ZJF
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Introduction

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. This service provides analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques.

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Principle Applications Procedure Materials

Principle

Isoelectric focusing (IEF) is a method of electrophoresis that separates proteins according to their isoelectric point. It performs in a particular pH gradient, in which proteins are separated by differences in their isoelectric points. The isoelectric point (pI) is the pH at which the net charge of the protein is zero. Protein is positively charged when its pI is above the solution's pH, so it migrates towards the cathode. Conversely, protein is negatively charged when its pI is below the solution's pH, so it migrates towards the anode. Protein has no net charge at its pl and stops migrating. Based on this principle, proteins can be separated into sharp bands with each protein positioned at a point in the pH gradient corresponding to its pI.

Applications

Food analysis

Procedure

1. Preparation: Prepare the gel containing the sample. Mix the solution and add it to a glass tube to form a gel. Prepare the fixing solution, dyeing solution and decolorization solution.
2. Electrophoresis: Add the appropriate acidic solution in the positive electrophoresis tank, and the appropriate alkaline solution in the negative electrophoresis tank. Switch on the electrophoresis apparatus and set the voltage and current. Perform pre-electrophoresis for a period of time to form a pH gradient in the gel. Then set the electrophoresis apparatus to a higher voltage and perform electrophoresis for a period of time.
3. Determination: Different protein components focus on the corresponding pI. Fix, stain and decolorize the gel. Finally, dry it for storage.