Cheese Please!
An Investigation into cheese using protein electrophoresis.
Prepared by J.Barfoot (Scottish Initiative for Biotechnology Education) in partnership with Science & Plants
for Schools (SAPS).
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 out 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, e.g. 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 weighs 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 and
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
gives cheeses their 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 microorganisms 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
NCBE 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 (but not set)
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
NCBE 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 into 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, just chip off a bit of the frozen cheese to use
each time.
6. Label small plastic tubes (microtubes) with cheese names.
7. Place the first sample in pestle and mortar (keep pestle and mortar on 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 to stop 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 NCBE 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 Can you hypothesise 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 affect 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 University 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.
stamped (first class) addressed envelope to:
The Scottish Initiative for Biotechnology Education
Darwin Building
King’s Buildings
Mayfield Rd
Edinburgh
EH9 3JR
Tel: 0131 650 7042, Fax: 0131 650 5371
Email: j.barfoot@ed.ac.uk
Send a large
Useful websites on cheese making:
http://cheesenet.wgx.com/ - Click on ‘The Library’ for some information on cheese
making.
www.anth.org.uk/hiramtrust/ - Click on ‘Articles (for downloading) and scroll down until
you see the one on cheese making. It downloads into a word document.
Advice and Technical Help:
Scottish Initiative for Biotechnology Education
Email: j.barfoot@ed.ac.uk Tel: 0131 650 7042
SAPS Biotechnology Scotland Project
Email: kath.crawford@sserc.org.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: