Extraction and

Characterization
of the Protein
Casein
SOSA
GADDI
Objectives
To isolate casein from milk
To be able to understand the methods
applied in the extraction of proteins
To apply spectrophotometric methods in
characterizing and quantifying extracted
casein and albumin
Possible Protein Sources
Plants
Invertebrates
Microorganisms
Casein
Primary protein in milk
General steps:
Protein released from cell by
homogenization
Membrane disruption by centrifugation or
fractional precipitation
Methodology
Extraction of Casein from Milk
Add 0.1 M HCl dropwise to 20 mL non-fat milk until
formation of a flocculent precipitate forms
Isolation of casein is achieved by acidification
because this would bring casein to its isoelectric
point so that, the extracting agent would react
with water instead of the protein being extracted

Decrease pH to about 4.8

Avoid adding excess HCl

Why is this so?
Addition of HCl and adjusting the pH to 4.8
facilitates:

better dissolution of Ca3(PO4)2 thus,

freeing casein from the cell for
precipitation
pH 4.8 is Caseins isoelectric point
Why avoid excess acid?
To much acidity would redissolve casein
because of its amphoteric nature

Effect:
lower yield of extracted Casein from non-
fat milk
Why non-fat milk?
For practicality purpose of preventing
proteins from precipitating with the
Casein thus,

Higher fat in milk, Lower % purity of Casein

ergo, Lower fat in milk, Higher % purity of
Casein
 Centrifuge the mixture
Separates Casein from other unwanted
cellular components that came from the
bursted cells
Separates cellular components according
to the density of the components and
density of the solution
Discard the supernatant
 Wash with 95% ethanol
precipitates and removes lactose in the
precipitate

Decant ethanol washings

Sample
must be washed until ethanol
washings is clear
Assures
that there is no more lactose present
in the precipitate
 Transfer precipitates into a watch glass

Wash precipitate with acetone

precipitates and removes lipids present in
the precipitate

Air dry under the fume hood

 Weigh dry crude casein extract

Prepare
20mL 1%(w/v) casein solution in
0.01M NaOH

Labelproperly and Keep it in the

refrigerator
Sample preparation

10
mL of 0.01% casein extract with 0.01M
NaOH was prepared as a stock solution
Protein Determination using
Warburg-Christian Assay
Absorbance of the casein extracted was
measured at 280nm and 260 nm

Use 0.01M NaOH as blank

Calculate the ratio of A280/A260 to

estimate the purity of the protein isolated
 In
calculating the protein concentration,
use the equation below:

Protein (mg/mL)= 1.55 A280 0.76 A260

Results and Discussion
What principles are involved in
the extraction of Casein?
Isolation of Casein from the cells
This is done by the process called
homogenization where-in cells are
ruptured to release its soluble proteins

This process also rupture other subcellular

organelles such as the mitochondria,
peroxisomes and the endoplasmic
reticulum, increasing unwanted materials in
the protein extract
 Differential Centrifugation
Since Casein is an insoluble protein, this
process would facilitate the separation of
unwanted molecular components based
on their densities that are not soluble in the
buffer used
The suspended particles are kept for further
purification and the supernatant is
discarded
Salting-out process
Works on the principle that: proteins tend to
be less soluble in concentrated salt
concentrations

Since no salt was used in this process, Ethanol

was utilized in order to salt-out the protein
casein from the solution

Salting-out occurs when ethanol reacts with

the water surrounding the protein to form
hydrogen bonding with it
 As
water is being attracted to ethanol,
Casein begins to interact with each other
forming hydrophobic bonds thus,
facilitating better precipitation

Multiple
washings with ethanol would
deprive the proteins from being hydrated
with water thus, becoming more insoluble
 Casein extraction chemical
equation
Ca2+Caseinate + 2HCl --------> Casein ( ) + CaCl2(aq) +
2H+

Other
acids may also be used in the acidification of
casein from milk as long as those acids DO NOT
precipitate Ca2+ ions.
Example of an acid that precipitates Ca2+ ions: H2CO3
Ca2+Caseinate + 2HCl --------> Casein ( ) + CaCl2(s) +
2H+
Addition of Acetone
Removes lipids present in the extracted
protein
Other methods of protein
extraction
Fractional Precipitation
Utilizes isoelectric precipitation where-in
pH variations of the solution alters the
state of ionization of the amino group and
carboxylic group of the protein

Solubility of protein is lowered to its pI

thus, protein-protein interaction becomes
dominant and that makes the protein
easier to salt-out
 This method also lowers the dielectric
constant of the solution thus, increasing
protein-protein interaction through
aggregation

This lowering achieved by the utilization

of miscible organic solvent to the solution
Ex: Hexane, Ethanol and Acetone
Summary of Fractional
Precipitation
Isoelectric precipitation
Via pH adjustment
Lowering of the dielectric constant
Via addition of a miscible
Temperature regulation
Lowering temperature as low as possible
Salt addition
Salting-in/ salting-out
Salting-in
Works
on the principle that: proteins tend
to be soluble in dilute salt concentrations

The
salt present in the solution would
screen of the protein from solvent thus,
precipitating out
The Warburg-Christian
Assay
Warburg-Christian Assay
The Warburg-Christian Assay utilizes the
absorption of proteins, specifically
tyrosine, tryptophan and phenylalanine at
a maximum absorbance of 280 nm while
the nucleic acid contaminants absorb
maximally at 260 nm
Warburg-Christian Assay
This
Assay utilizes the equation below:
Protein (mg/mL)= 1.55A280-0.76A260
Nucleic acids, contaminants to the
protein extracted, absorbs 10 times
greater than proteins, they absorb
maximally at 260 nm and absorbs greater
than at 280 nm which poses a
disadvantage for this assay but
Warburg Assay
But the equation previously shown
corrects the interference caused by the
nucleic acids by coming up with a
correction factor for the abosrbancies at
280 and 260 where in the maximal
absorption of nucleic acids at 260 nm and
of proteins at 280 nm are correlated
Warburg-Assay
This Assay can detect 20-3000 ug/mL of
protein
Results

Protein concentration (mg/mL)=1.55A2800.76

A260
Protein concentration (mg/
mL)=1.55(0.094)0.76(0.100)
Protein concentration (mg/mL)=0.14570.076
Protein concentration (mg/mL)=0.0697~ 0.070
mg/mL
Percent purity
Casein
A280/A260= (0.094/0.100)= 0.94

100-4.0= 96%
Possible Sources of Errors
Parallax errors in measuring reagents
Human errors in following the instructions
of the experiment
Contamination of other cellular
components that were present in the
solution upon reading its absorbance in
the UV-VIS