Post-laboratory Report on

Exercise 4.1
Isolation of Proteins

Vikki Anne R. Cedo

CHEM 160.1 - 3L
2nd Semester 2014-2015

Groupmates:
Desiree Joy Cerico
Ma. Kriselle Ornales
Mary Ranzelle Pasang

Ms. Korina Vida G. Sinad

Laboratory Instructor

Exercise 4.1
Isolation of Proteins
Proteins are by far the most important of all biological compounds. Many
types of this biological compound exist, and they perform a variety of functions
including structure, transport/movement, hormones, protection, storage and
regulation. This diversity of functions make them the most studied biological
compound. Many isolation and purification techniques were successfully done by
scientists as well as techniques for determining the quality and quantity of the
isolate.
Making use of the knowledge of solubility and isoelectric point of proteins,
protein precipitation method was established. Proteins are isolated from a mixture
of soluble substances. Knowing the solubility of your protein of interest, you can
now easily precipitate out your protein from the solution by designing a
precipitation technique (choosing what reagent you'll add to the solution). Salting
out process and isoelectric precipitation are the two most commonly used
techniques.
Salting out is a technique wherein salts such as ammonium sulfate or sodium
sulfate are used to precipitate out proteins by adding them into the solution.
Ammonium sulfate is the one which is widely used for protein precipitation by
salting out because it is highly soluble, available in the highest purity level and does
not change the pH to extremes and it does not denature proteins; it also protects
solution from bacterial growth. During ammonium sulfate precipitation, the salt has
to be added in small amounts with constant stirring so as to avoid accumulating
high concentration of salts. When large amount of salt is added to an aqueous
solution of proteins, the salt will require more amount of water for its dissolution.
This will lead to competition for water molecules then protein hence addition of salts
take up water molecule from proteins. The ionic interactions between water
molecules and protein are reduced and as a result hydrophobic interactions
dominate. The hydrophobic amino acid patches present in all the proteins attract
each other and form aggregates. These aggregates are nothing but the proteins in
the form of precipitates.
Isoelectric precipitation is another protein isolation technique. This technique
makes use of the knowledge of the pH of the solution and the IpH or isoelectric
point of the protein you desire to isolate. Changes in pH of the solution change the
ionization state of the proteins' functional groups and its net charge. When the pH
of the solution is equal to the isoelectric point of the protein, it is the time when the
protein becomes least soluble and therefore precipitates out of the solution. The
solubility of the protein increases if the pH of the solution is somewhere to the right
or to the left (higher or lower) of the isoelectric point.

Protein solubility depends on numerous factors. It is observed that at low

concentration of the salt, the solubility of the protein increases by a small
percentage. This phenomenon is called salting in. At high concentrations of the salt,
the solubility of the proteins drop dramatically and this is called salting out because
the proteins precipitate out of the solution. In salt precipitation, the anions appear
to be more important. pH, temperature and protein concentration affect ammonium
sulfate precipitation of proteins to large extent. Higher ammonium sulfate is
required for precipitating highly soluble proteins.
Ammonium sulfate can be used for precipitation of total proteins at ~90%
saturation or for differential precipitation level of proteins using different saturation
of salts. Up to 20 % saturation, ammonium sulfate precipitate particulate materials,
and preaggregated and very high molecular weight proteins and at 90 % saturation
it precipitates almost all proteins.

Table 4.1.1. Preparation of crude egg albumin by ammonium sulfate precipitation.

Steps
Filtered undiluted egg white
Addition of 1 M acetic acid
Removal of precipitate| Precipitate:
| Filtrate:
After addition of 7.26 g ammonium
sulfate powder
Filtration
| Residue:
| Filtrate:
After addition of 3.9 g ammonium
sulfate powder
After 30 minutes of standing
Filtration
| Residue
| Filtrate
After air drying

Observations
Yellowish viscous liquid
Formation of white clumps
White viscous clumps
Yellowish opaque liquid
Formation of bubbles, cloudy yellow
liquid
White bubbles with white precipitate
White cloudy liquid
White, milky liquid with bubbles
Yellowish liquid
White bubbles with white precipitate
White, milky liquid
White precipitate

Mass of filter paper: 1.707 g

Mass of filter paper + crude egg albumin: 3.397 g
Mass of crude egg albumin: 1.69 g

Increasing the concentration of ammonium sulfate in the experiment to

obtain or isolate proteins is essential because the higher the concentration of the
salt, the more proteins that it will be able to precipitate out of the solution. In other

words, the higher the ammonium sulfate concentration (salt), the least soluble the
proteins become in the solution, causing them to precipitate out.

Table 4.1.2. Isoelectric precipitation of casein.

Test tube no.
1
2
3

pH
2.7
4.7
6.7

Observations
Clear solution
Cloudy
Clear solution

Calculation of yield (60% ammonium sulfate precipitate):

volume of egg
(weight of dri ed precipitate)

3.397 g
40 mL

= 0.084925 x 100 = 8.4925 or 8.50%

In the isoelectric precipitation of casein, only test tube number 2 was

observed to have a cloudy mixture and the other two test tubes contain clear
solutions. Test tube number 2 contains acetate buffer and has a pH of 4.7, casein,
distilled water and 1M NaOH and 1M CH 3COOH, while test tube number 1 contains
glycine-HCl buffer casein, distilled water, 1M NaOH and 1M CH 3COOH with a pH of
2.7and test tube 3 contains phosphate buffer, casein, distilled water, 1M NaOH and
1M CH3COOH with a pH of 6.7. After adding the solution of casein, distilled water,
1M NaOH and 1M CH3COOH to the buffers, only test tube number 2 was observed to
exhibit cloudiness. It can be concluded that the IpH of casein is 4.7 since it where
the mixture got cloudy. At pH 2.7 and 4.7 (test tubes 1 and 3), it can be concluded
that casein is soluble because pH 2.7 is lower than its isoelectric point and 6.7 is
higher than the isoelectric point. The theoretical IpH of casein is 4.6. It is the pH at
which the protein is least soluble. Test tube no. 2. exhibited a cloudy mixture so it
can be concluded from the result of the experiment that casein's IpH is at 4.7.
Casein precipitated out of the solution because its solubility is decreased at that pH,
so they aggregated together and precipitated out.

References:
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry.
Brooks/Cole by Thomson Learning.
Brown, C. (2005). Introduction to Biochemistry. McGraw-Hill International.

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