Cheese Please!

An investigation into cheese using protein

electrophoresis
Prepared by J.Barfoot (Scottish Initiative for Biotechnology Education) in partnership with SAPS
(Science and Plants for Schools)
Protein electrophoresis - Cheese Please!
Background information
Please consult the NCBE/SAPS ‘Protein Power!’ Teacher and Student guides
for detailed background and technical information on proteins and protein
electrophoresis.

Electrophoresis
Electrophoresis is a technique that uses electricity to separate out molecules.
In this case we are using electrophoresis to separate proteins on an agarose
gel. Large proteins move slowly through the gel and small proteins move
more quickly, therefore the proteins are separated according to their size.

Proteins
There are many different types of proteins. For example, structural proteins
such as the proteins found in hair and skin, catalytic proteins such as
enzymes and messenger proteins which act as signalling molecules, for
example hormones. Proteins are formed as long chains of amino acids, the
order of the amino acids in the protein is determined by a specific DNA
sequence, or gene.

Protein structure
Proteins do not remain in long amino acid chains (known as the primary
structure). The chemical composition of amino acids in the chain causes
bonds to form between the molecules. This causes the chain of amino acids
to fold and coil, resulting in a protein with a 3D shape. This is called the
tertiary protein structure. In the following practical experiment, one of the
steps involves boiling the proteins in a specific buffer (Laemmli buffer). This
results in the denaturing (or unfolding) of the proteins back to their primary
structure. All proteins of the same size will run to the same part of the
agarose gel. This is what forms the bands.

The molecular weight of proteins

Different proteins are different sizes (or molecular weights). These weights
are so tiny that we cannot measure them in grams or even nanograms. The
unit for the molecular weight of proteins is a dalton. A dalton is defined as an
atomic mass unit approximately equal to the mass of a hydrogen atom. That
basically means that if a protein has a molecular weight of 6000 daltons it
weights about the same as 6000 hydrogen atoms. The protein standards
used in this experiment provide examples of proteins of a specific molecular
weight (in daltons). You can use these standards to estimate the sizes of
protein bands in your gels.
How is cheese made?
The basics of cheese-making involves three steps:

1. Cause the milk to coagulate (change from a liquid to a solid) into curds.
This can be done by various methods
o by the addition of an acid such as vinegar or lemon juice.
o by the addition of a bacterial culture (which can metabolise
lactose) which produces lactic acid.
o by the addition of rennet, which contains the enzyme rennin.
2. Separate the coagulated curds from the remaining fluid, called whey.
3. Ripen cheese (if required).
4. Eat.

To make soft cheese steps 1 to 3 can take from an hour to a few days, for
hard cheese steps 1 to 3 can take months. This basic method has many
variations and this is what makes cheeses have different flavours and
textures.

The simplest cheeses such as cottage cheese are made this standard way
without any maturing. However some cheeses are matured for different times
at different temperatures to generate different flavours and textures, naturally
occurring micro-organisms play a huge part in this maturing process. Blue
veined cheeses are inoculated with a Penicilium spore which creates a
particular flavour and aroma. Brie and Camembert have their surfaces
inoculated with another type of Penicilium spore, this adds a particular flavour
to the cheese and creates the white skin on the outside of the cheese.

This project investigates the differences in cheese using protein

electrophoresis.

Technical information
Equipment and materials

NBCE protein electrophoresis kit (Contains everything your need to do protein

electrophoresis e.g tanks, buffers, stain, electrodes, plastic tubes)
Batteries
Cheese
Water bath at 100°C
Pestle and Mortar
Two plastic boxes containing crushed ice – Don’t forget to make ice in advance.
Distilled water
Tube containing powdered protein standards.

Preparation of materials
The NCBE kit has detailed instructions for preparation of materials. It can be
helpful to make your agarose gel in advance.
Preparing the 3% agarose gel
1. Weigh out 1.5g of agarose into glass beaker or bottle.

2. Add 50cm3 of 1X TB Buffer.

3. Place in boiling water bath until agarose has melted – swirl

occasionally to ensure that agarose is thoroughly mixed.

4. Allow gel to cool

5. Place gel tank on flat surface and fit the 6 toothed comb into the slot at
the end of the gel tank. Ensure comb is pushed all the way down.

6. Pour the molten agarose into the gel tank, 50 cm3 of 3% agarose
should be enough to make up 4 or 5 gels.
NB. The thinner the gel the better you will be able to see your protein bands. Agarose is
very expensive so it pays to be frugal. However make sure you have covered the tips of
the comb otherwise you will have nothing to load your protein sample into.

7. Leave the tank undisturbed for 15 – 20 minutes until the gel sets.
NB. Gels can be made in advance and stored in the plastic bags (containing a little
TB/SDS buffer) in the fridge for up to a week.

Preparing protein standard solution

NB this solution must be made up fresh every time
1. Add 1cm3 of distilled water to tube containing protein standard
powders.
2. Mix well for a few minutes
3. Keep in ice.
Starter experiment
Materials
NBCE protein electrophoresis kit
Batteries
5 types of cheese to be investigated
Powdered protein standards
Water bath at 100°C (can substitute this with freshly boiled water from a kettle)
Pestle and Mortar
Two plastic boxes containing crushed ice
Floating tube holder
Plastic 1cm3 pipettes

Instructions
These instructions are slightly different from the NCBE instructions, it is hoped
that the extra steps will help you get better banding patterns.
NB protein degrades easily through the action of proteases, the cooler you keep your sample
and less time your protein sample sits around the better banding patterns you will get.

1. Pour 3% agarose gel and leave to set.

2. Place pestle and mortar on crushed ice to cool
3. Place 6cm3 of TB/SDS Buffer in a small beaker and place in crushed
ice to cool.
4. Make up protein standard solution and place on ice.
5. Collect a sample about the size of a grain of rice of each cheese to be
investigated.
NB the cheeses can be kept frozen in the freezer. Don’t defrost them each time, just chip off a bit of
the frozen cheese to use each time.
6. Label small plastic tubes (microtubes) with cheese names.
7. Place first sample in pestle and mortar (keep pestle and mortar in ice)
and add 1cm3 of the cooled TB/SDS Buffer.
8. Grind sample in buffer.
9. Using a clean plastic pipette remove cheesy solution from pestle and
mortar and place in a labelled clean tube.
10. Place microtube on ice.
11. Wash pestle and mortar in cold water and dry.
12. Repeat steps 6 – 10 for each of your samples.
13. Label a second set of microtubes with the five cheese names and an
additional microtube with PS for Protein Standard. Into each of these
microtubes add 0.25 cm3 of the Laemmli Buffer.
 14. Add 0.25cm3 of your cheese samples (avoid the ‘bits’ that sink to the
bottom) and the Protein Standard Solution to the relevant labelled
tubes containing Laemmli buffer.
NB Change pipettes each time to avoid contamination between the different samples.
15. Place the tubes in boiling water for 5 minutes and place on ice.
NB Use a pin to put a hole in the lid of the tubes it stops them popping open.
16. Pour slightly more than 10cm3 of TB/SDS Buffer onto the set gel.
17. Load your five cheese samples and the protein standard into the gel
carefully (NCBE Student guidelines are excellent for this step).
NB If it is helpful you can use the sheet provided to record the order in which you loaded
your samples.
18. ‘Run’ the gel (as explained in the NBCE Student guidelines).
19. Stain the proteins (again follow the NCBE Student guidelines).
20. Compare the cheese protein bands with the protein standard bands.

Recording your results:

Once the gels have thoroughly destained you will want to record your results.
A few options:
o Carefully measure the bands on the gel and create a copy in your lab
book with the exact same measurements.
o Photograph the gels on a light box (normal or digital camera)
o If you have access to a computer scanner, you can scan the gels.
Cover them in cling film so that you do not leave blue stain on the
scanner.

Analysis questions:
o Use the protein standard bands to approximate the sizes (in daltons) of
the protein bands in the different cheeses.
o Are there differences in the protein banding patterns (fingerprints)
between the cheeses?
o Can you deduce what specific proteins any of the bands may
represent?
o Hypothesis what any extra band may be caused by?
Possible project titles
Investigate the protein bands from cheeses from different animals. Is it
possible to tell which animal a cheese was made from simply by the protein
bands?

Compare the proteins in soft cheeses to hard cheeses. Are there any
differences? Can these differences be related to the different way these
cheeses are made?
Mature cheeses have a much stronger flavour than mild cheeses. Compare
cheese of different ages. What happens to the proteins as a cheese ripens?

Some cheeses are made by inoculation with a particular mould to add flavour
(e.g. Roquefort is made with Penicillium roqueforti). Compare Blue cheeses
with non blue cheeses. How might a mould effect the proteins in the cheese?

Some cheeses (e.g stilton) are flavoured with walnuts, apricots, blueberries,
ginger etc. Can you detect the presence of these ‘extra’ proteins on the
protein gels?

Resources
The NCBE Protein Power kit can be obtained from:
The National Centre for Biotechnology Education
The Univeristy of Reading
Whiteknights
PO Box 226
Reading
RG6 6AP
Tel: 0118 9873743, Fax: 0118 9750140
Email: NCBE@reading.ac.uk
Web site: www.ncbe.reading.ac.uk

The protein standards can be obtained free from the address below. Send a
large stamped (first class) addressed envelope to:

Scottish Initiative for Biotechnology Education

Darwin Building, King’s Buildings
Mayfield Rd
Edinburgh, EH9 3JR
Tel: 0131 650 7042, Fax: 0131 650 5371
Email: SIBE@ed.ac.uk

Advice and Technical Help:

Scottish Initiative for Biotechnology Education
Email: SIBE@ed.ac.uk
Record Sheet
Date:

Aim of Experiment:

Cheese used:

Notes on practical work e.g. did anything go wrong? Did I forget a step?:

Layout of Gel:

Results:

Comments, Ideas for next time: