Determination of isoelectric point of protein (casein).

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Determination of isoelectric point of protein (casein).

Introduction:

Casein is a globular colloidal protein.  Globular proteins are hydrophobic proteins which in certain external condition are soluble in eater.

The ph at which the protein is electrically neutral is known as the isoelectric point.  A globular protein such as a casein becomes increasingly insoluble as it approaches its isoelectric point.

Objectives

The object of this experiment is to determine the isoelectric point of casein (protein), which can be precipitated from the solution.

Apparatus

9 test tubes

pipettes

  • 1ml
  • 5ml
  • 10ml

colorimeter

Materials

Distilled water

Acetic acid

  • 0.01 M
  • 0.1M
  • 1.0 M

casein – 0.5g/1 in 0.1 M sodium acetate

Method

  1. The calorimeter is switched ON to allow it to “warm up”.
  2. In order to distinguish between the different acidity levels contained in each test tube, the 9 test tubes were labelled from 1-9.  This is important because all solution are a similar colour.
  3. Following the designated volumes required on table 1, the volumes of distilled water was then pipetted into each test tube.  The acetic acid was then pipetted into each test tube according to the values in the table 1.
  4. In order to reduce the chances of contamination , the designated amounts of 0.01M acetic acid was pipetted first to test tubes 1 and 2 because the 0.01 M acetic acid  is the least concentrated acid of the 3 acids.  If the process was carries out the other way round, the higher concentration of the 1.0 M acid would raise the acidity level of the 0/01 acid.
  5. Second, the designated amounts of 1.0 M acetic acid according to table 1 was added to test tubes 3,4,5,6,7 and 8.
  6. Third, the designated amount of 1.0 acetic acid according to table 1 was added to test tube 9.
  7. Fourth, 1ml of casein 0.5g/1 in 0.1M sodium acetate was pipetted into each test tube.  The pipette was then quickly blown out each time as much of the casein enters the test tube each time.  It is important to ensure as accurate a measurement as possible by reading off the bottom of the meniscus precisely each time because the volume and concentration of casein added to each test tube has to remain constant.
  8. Then covering each test tube with a different finger for each solution in order to avoid contamination, the solution contained in each test tube were shaken together.
  9. After 30 min, the degree of turbidity was recorded on a scale of 0 to 3+ and recorded in a table 1.
  10. In order to measure the turbidity of each solution by using the calorimeter, the 9 samples from each of the test tubes were poured into 9 curvettes destined for the calorimeter and labelled accordingly.
  11. One curvette was filled with distilled water  which acts as a marker for zero turbidity.
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  1. The curvette containing the distilled water was placed into the calorimeter and the value of turbidity  ( light absorbency)  was recorded for each sample.  The calorimeter was set to zero according to the clarity of the distilled water.
  2. The remaining 9 curvettes were each placed individually into the calorimeter and the value of turbidity  ( light absorbency)  was recorded for each sample.  The calorimeter was reset to zero using the curvette containing the distilled water for each sample to maximise accuracy.

Observations and Conclusion

When acetic acid was added to the distilled water, the ...

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