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Isolation of casein from milk experiment and results + discussion and conclusion
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Isolation of casein from milkexperiment results
written by:
Sonia Patricia
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TEST REPORT
Title: Isolation of Casein from milk
Dates: 16-11-2012, 30-11-2012
Names: Elena van Zalen and Sonia Stoica
Aim: The aim of this experiment is to isolate casein from skimmed milk in order to determine its
concentration based on the dry weight, (% m/V) also to find the yield based on the Bio-Rad Protein
Assay method( % m/V) and to analyze the composition of the isolated protein mixture by means of a
SDS polyacrylamide gel electrophoresis ,as well as to determine the mol mass of the casein-proteins
based on the calibration standards.
Protocol: BCT EXP003 Isolation of casein from milk
Deviations from the protocol :Instead of the buffer A we used demineralized water.
Principle:
Normal bovine milk contains 30–35 grams of protein per liter of which about 80% is arranged in
casein proteins, in weight. Compared to the other proteins in milk, casein has got a larger molecular
weight and a larger structure. Most of the casein proteins are bound into the micelles. Each casein
micelle is roughly spherical and about a tenth of a micrometer across. There are several competing
theories regarding the precise structure of the micelles, but they share one important feature: the
outermost layer consists of strands of one type of protein, k-casein, reaching out from the body of
the micelle into the surrounding fluid. There are four different types of casein proteins: αs1-, αs2-, β-,
and κ-caseins. These kappa-casein molecules all have a negative electrical charge and therefore repel
each other, keeping the micelles separated under normal conditions and in a
stable colloidal suspension in the water-based surrounding fluid.
Milk contains dozens of other types of proteins beside the caseins including enzymes. These other
proteins are more water-soluble than the caseins and do not form larger structures. Because other
proteins are more soluble and they do not form larger structures than the casein proteins, they get in
the top layer of the centrifuge tube during centrifuging. As is described above, casein is a larger
molecule and therefore has a bigger mass. This means that casein will get on the bottom of the
centrifuge tube during centrifuging.
To check to amount of protein that are present in the two pellets and the supernatant, a
spectrophotometric protein determination is carried out with, for example, Bio-Rad Protein Assay. By
making a calibration line and then measure the samples at 595 nm, the protein concentrations can
be determined.
With SDS polyacrylamide gel electrophoresis (SDS-PAGE) proteins can be separated with a mass
of approx. 10 K to 150 kDa (Kilo Dalton, 1 kDa = 1000 g/mole.The protein molecules are
separated according to molecular size
An electric field is applied across the gel, causing the negatively-charged proteins to migrate across
the gel towards the positive (+) electrode (anode). Depending on their size, each protein will move
differently through the gel matrix: short proteins will more easily fit through the pores in the gel,
while larger ones will have more difficulty. After a set amount of time, which in this case is about an
hour, the proteins will have differentially migrated based on their size; smaller proteins will have
traveled farther down the gel, while larger ones will have remained closer to the point of origin, like
described before. Proteins may therefore be separated roughly according to size (and thus molecular
weight); however certain glycoproteins behave anomalously on SDS gels.
Mode of operation: see protocol
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Pre-laboratory preparations
1. Look up the weight percentage of casein in milk. State what kind of milk you are going
to use and why.
Theoretical weight percentage of Caseïn: 76–86%. The milk we are going to use is skimmed milk,
because of the lower fat concentration. And a maybe higher concentration of proteins.
2. Look up all the R and S sentences of the chemicals needed. If necessary, use the
protections means needed (gloves; acid fume chamber).
HCl
R-phrases
R34- Causes burns
R37- Irritating to the respiratory system.
S-phrases
S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
S36 Wear suitable protective clothing.
S45 In case of accident or if you feel unwell, seek medical advice immediately (show label where
possible).
3.Find out what type of protein casein it is. Predict the results by means of the theory
you found.
It is a phosphor-protein because it tis a protein from a mammal and milk is a mammal product. It will
probably have a high yield. The isoelectric point of casein is 4.6. Since milk's pH is 6.6, casein has a
negative charge in milk. Because the casein is negatively charged, it will travel down faster the
electrophoresis gel.
4. Look up what the working of gel electrophoresis is based on
Electrophoresis is how the proteins are pushed through the gel filter by applying an electrical current.
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Observations and Results:
Table 1: Mass and concentration of casein protein in milk, based on dry weight.
Wet mass g % dry mass Dry mass Volume (ml) c (mg/ml)
P1 1.57 0.628 52 12.08
P2 2.0 40 0.800 120 6.60
Table 2 : Concentration of casein in milk sample, based on Bio-Rad Protein Assay
Absorbance Dilution factor c( mg/ml )
0.2mg/ml standard 1 0.490
0.4mg/ml standard2 0.595
0.6mg/ml standard3 0.857
0.9mg/ml standard4 1.012
SN2 0.599 5x 1.735
P1 0.939 2x 1.560
P2 0.432 10x 1.350
Using the formula from the graph below (fig1) the concentration of the samples could be calculated(
also taking into account the dilution factor SN2 solution: 5x, P2 solution: 10x, P1 solution: 2x )
Calibration line of the standards
y = 0.7856x + 0.3261
1.20
1.00
0.80
Absorbance
0.60
0.40
0.20
0.00
0.00 0.20 0.40 0.60 0.80 1.00
c (mg/ml)
Fig 1: Calibration line of the standards based on absorption at a certain concentration
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