Protein isoelectrelectring.

, objective: to understand
the meaning of isoelectrelectrists and its relationship
the
of proteins and molecules.II, principle:
protein molecular weight is very large, it can form a stable and equal solution, mainly because protein molecules have the same symbol of the charge, while the protein molecules around a solventized water film, to avoid protein molecules between the accumulation and subside.Protein molecules have a lot to do with the pH of the solution, the protein is a sex electrolyte, the protein in the alkaline solution into anions, in the acidic solution into cations:
protein molecules with zero net charge pH is called the protein's isoelectric point (PI). At isoelectrients, protein molecules do not move to any pole in the electric field, and the tendency of fusion between molecules and molecules due to collision increases, so the viscosity and permeation pressure of the protein solution can be reduced to a minimum, and the solution becomes cloudy. If a certain amount of solvents such as ethanol and acetone are added, they compete with protein molecules for water molecules, trying to reduce the thickness of the protein hydration layer and make turbidity more obvious.
all kinds of protein isoelectric points are not the same, but more acidic, such as casein in cow's milk is 4.7 to 4.8, hemoglobin and other electrical points are 6.7 to 6.8, insulin is 5.3 to 5.4, fish protein is a typical alkaline protein, its isoelectric point in pH12.0 to 12.4. In recent years, the isoelectrment points of proteins can be accurately determined by the use of isoelectrelectration focusing technology, but certain experimental conditions are required. This experiment uses the turbidity formed by the protein in different pH solutions to determine, that is, the pH value at the maximum turbidity is the iso-point value of the protein, although this method is not very accurate, but under general experimental conditions can be carried out, the operation is also simple.


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, operation steps:
1. Prepare the protein gel
(1) called take 3 grams of casein, put in
beaker
, add 40 degrees C distilled water.
(2) add 50ml 1 mol. L
-1
sodium hydroxide solution, stirred over a slight heat until the protein is completely dissolved. Transfer the dissolved protein solution to a 500 ml
capacity bottle
and wash the bea cup with a small amount of distilled water, pouring into the capacity bottle.
(3) add another 1 mol in the capacity bottle. L-1 acetic acid solution 50 ml, shake well.
(4) add distilled water to 500 ml, get slightly cloudy, at 0.1mol. Casein collate in L-1 NaAC solution.
2. Isoelectric point determination
the following table order in each tube to add protein glue, and accurately add distilled water and various concentrations of acetic acid solution, immediately after the addition of shake.the turbidity produced by the tubes and determine the isoelectrin ejectors based on the turbidity. When observing, it can be used to indicate turbidity. .