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° (can substitute this with freshly boiled water from a kettle) C 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:

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