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Isoelectric Precipitation of Proteins: Casein from Milk


Materials required:


1)  Raw milk - 100ml

2)  0.2N HCl - 50ml

3)  Diethyl ether - 50ml

4)  50% Ethanol - 50ml

5)  Whatman No 1 filter paper strip (Size 25×50mm) - 2 no.




  1. Measure 100ml of milk in a measuring cylinder and transfer 25ml of milk to four Oakridge centrifuge tubes each.
  2. Centrifuge the milk in a centrifuge at 4000rpm at room temperature (25- 30o C) for 20 minutes. This is done to remove the fats and lipids from the mixture.
  3. After centrifugation, carefully remove the fats and lipids from the surface of the milk with a spatula.
  4. Then transfer the milk from all the tubes into a beaker and add equal volume of distilled water and stir well. Now check the pH.
  5. Start adding 0.2N HCl drop by drop into the milk mixture and stir well.
  6. Note the PH at which precipitation (white curdy substances) appears. The pH should be 4.6.
  7. Take the curdy precipitate and allow it to sediment.
  8. Now decant the supernatant using a filter paper and funnel and wash the precipitate with distilled water to remove the salts, then wash with diethyl ether and ethanol.
  9. Dry the precipitate and take the weight of the casein and record it.


Differences Encountered In a Real Laboratory:

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab


  1. Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the exhaust fan  and make sure that all the reagents required for the experiment are  available. If they are not available, prepare the reagents using the components for reagent preparation.
  2. Make sure that the milk you are using is not skimmed milk.  Commercially available packet milk will be skimmed, so the fat content will not be present, and you will not have to do the centrifugation part of the experiment.
  3. Care should be taken while handling caustic acids like Conc. Hydrochloric acid [HCl]. These acids should be prepared in FUMEHOOD only. However, the prepared 0.2N HCl can be opened and used on the lab bench  as it is weakly acidic.
  4. Calibrate the pH meter with standard buffer before you start to do the experiment. Make sure that the pH meter is working properly.
  5. Wash the pH electrode thoroughly with distilled water and wipe the electrode smoothly. Care should be taken to ensure that lint free tissue paper is used to wipe the pH electrode.
  6. Do not immerse the electrode into the solution deeper than 20mm.
  7. There will be fluctuations in pH reading when the electrode is dipped into the solution, so make sure to wait long enough for the reading to stabilize.
  8. Once the reading has been taken, immediately remove the electrode from the sample and rinse properly with distilled water.
  9. Decanting the supernatant can be done using filter paper or a cheese cloth. Filter paper may take a longer time to decant.
  10. Cautiously use Diethyl ether as it is highly volatile and flammable. Do not allow diethyl ether to come in contact with hot surfaces.
  11. Make sure that the pH electrode is properly dipped in the storage solution after  completion of the experiment.
  12. When you leave the lab, make sure to switch off the exhaust fan.





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