Science Buddies: "Ask an Expert"

My science fair project is Comparing Levels of Carcinogens in Grilled Chicken Prepared by Different Types of Charcoal
I have heard of the Ames Test to detect mutagenic activity, but are there other ways to detect the carcinogens? Are there any other processes that involve BIO-RAD's protein electrophoresis or a way to incorporate this process? -it is okay if I restructure my project so any advice is accepted.

Hi akamai.t.ramos,

The ames test is actually the most widely accepted test for carcinogens. If you want to look at proteins (and use protein electrophoresis), you can always try to see if certain genes (expression) in yeast are affected by the carcinogens from different charcoals.

Hope that helps!

-Sam

Hi,

This is a great project idea. What information have you found in your background reading so far? Is there a particular carcinogen you are interested in investigating? Here is a news report about one specific carcinogen, phenylmethylimidazopyridine, or PHIP, a heterocyclic amine that can be formed when chicken is exposed to high temperatures:

http://www.medicalnewstoday.com/releases/122937.php

Here is an article that describes an analytical technique for heterocyclic aromatic amines based on high pressure liquid chromatography and mass spectrometry. This is very specialized equipment and the column used for this type of analysis is very expensive, so you would need access to a laboratory and someone willing to train you to use the equipment.

http://ntr.oxfordjournals.org/content/13/2/120.abstract

Here is another paper that gives a detailed protocol, also using an HPLC technique for quantitating heterocyclic amines in beef:

https://www.llnl.gov/str/pdfs/UCRL-JC-116450.pdf

I suppose you could also do a standard mutagenesis assay.

http://en.wikipedia.org/wiki/Ames_test

I am not familiar with any electrophoresis techniques for this type of analysis. The carcinogens are small molecules and electrophoresis is more suitable for analysis of larger molecules.


Donna Hardy

sk1 wrote:Hi akamai.t.ramos,

The ames test is actually the most widely accepted test for carcinogens. If you want to look at proteins (and use protein electrophoresis), you can always try to see if certain genes (expression) in yeast are affected by the carcinogens from different charcoals.

Hope that helps!

-Sam

Sam,

Mahalo Nui! Thank you so much for your advice.

Tiana

Ms. Donna,

I did actually choose to investigate PhIP, there were other too like in the KFC study, but since PhIP was of more quantity then I chose that one.

Thanks for the protocol for detecting carcinogens in beef! This will be very beneficial to my project for I will go along their methods but with chicken instead.

What do you suggest that I do? The Ames Test or the chromatography process? My mentor at my school said she is able to order the chromatography kit from BIO-RAD and she will assist me. It's a bit pricy but she said she's able to purchase it.

Mahalo Nui,
-Tiana

Also, when using the chromotography protocol, which of these different protocols would I use?

http://www.molecularstation.com/protoco ... protocols/

Or, out of all the three ways to detect the carcinogens, which would be the easiest do you think? or more effective?

Hi Tiana,

Which kit from Bio-Rad is your teacher considering? I am not familiar with any test kits that are suitable for this specific application

The Ames mutagenicity test is probably the most standardized method and is a general all-purpose assay. The HPLC technique would be useful and very specific, but would not work if the specific carcinogen you are looking for is not present in the sample. The advantage of HPLC is that you could quantitate the carcinogen. So I guess it depends on the objective of your project. You would learn a lot with either project.

Let me know the catalog number for the Bio-Rad kit and I'll give you my opinion on that option.


Donna Hardy

Ms. Donna,

Here's the product info:
Size Exclusion Chromatography Kit
166-0008EDU

There are other chromatography products they have but some of them I am not familiar with or think that it would detect such a small substance like an amine.. The problem with the Size Exclusion Chromatography Kit is that in the catalogue it implies that the amines wouldn't be able to be detected, so I'm iffy about that.

The problem with the Ames Test is that my mentor doesn't have the materials for it. She helped me find some other staff members at UH at Manoa & some at JABSOM, so I'm hoping that someone will reply back to me.

If I'm not able to get a mentor that would assist me with the Ames Test or the chromatographic tests, I have to find a simpler way on conducting the experiment where I can stay with the mentor I have now. I looked at the references on the back of the article you gave me about the microwaveable fried beef patties & there is an article I'm going to look more into titled "Simple methods for quantifying mutagenic heterocyclic aromatic amines in food products."

If at all else fails, I'm going to have to change to a more feasible project.

Mahalo Nui,
-Tiana

Here's another protocol that I found for detecting carcinogens:

http://books.google.com/books?id=dDtXkw ... ns&f=false

Hi,

The Bio-Rad chromatography kit is a demonstration of size exclusion chromatography and would be useful for separating a mixture of proteins based on their molecular weight. It would not give you results that are specific for detecting carcinogens. Chicken and beef samples contain a mixture of proteins and I’m not sure that the column provided in this test kit would resolve them. I would not recommend that you purchase this kit for your project.

The yeast DEL assay that you found is somewhat similar to the Ames test and required a specific strain of yeast, a growth medium, individual amino acids, and other specialized materials Did you read through the materials and method section? Does your mentor have these materials available? They are very specialized.

If your mentor does not have the material for the Ames or the yeast DEL test, then you do need to find a different project. It’s always a challenge to match great ideas with experiments that are doable. What types of materials does your mentor have available to use? Are there any other projects that you are interested in doing?

Donna Hardy

Ms. Donna,

I have showed my mentor this article and she does not have the materials. If the people do not get back to me in time, then I am going to change my project. My mentor right now has materials to run protein gels. She knows all about that and has the equipment necessary to do it. If I was going to change my project, I would go somewhere along those lines? -with proteins, and running them through gels-

Do you have any ideas on projects that I could do based on proteins? Maybe instead of carcinogens I could do bacteria?

Mahalo Nui,
-Tiana

Hi,

You have great resources available! With the protein electrophoresis gels, you could do a protein purification project. If you used the Bio-Rad chromatography kit, for example, you do gel electrophoresis on the green fluorescent protein to check the purity. For an original science fair project, however, it might be good to pick a protein that would have more significance or a practical application. Why don’t you do ask your mentor and Google search to look for a protein to purify? For example, perhaps you could purify an enzyme that would have an industrial application. Or perhaps one of the proteins from milk. I’m sure there are lots of possibilities.

Does your mentor also have any chromatography products that you could use?

I will try to think of some other ideas as well.

Donna Hardy

Ms. Donna,

Okay! That's a reliever. I could do more research on purifying proteins and see what comes up. I'm also in the Health Academy so something that could be health-based/related would be a cherry on top. I'll look more into the different proteins too.

As for the chromatography products; my mentor doesn't have it but the product that I mentioned earlier, the BIO-RAD Size Exclusion Chromatography Kit, we are able to purchase it.

Thanks for all your help,
-Tiana

Hi,

What type of research projects are currently being done at the Health Academy? Are there any protein-containing samples available for you to test? What is your mentor doing with the electrophoresis gels? Is there anyone cloning recombinant proteins at the Academy? It would be ideal if you could do a side project and some experiments that would be an extension of an ongoing project. Does this help you identify a possible topic for your project?

Donna Hardy

Ms. Donna,

Well another person that my teacher is co-mentoring is doing his project on testosterone depletion in castrated and non-castrated carcass. Another person is doing effects on epidurol, & another is in a lab doing "Using a Microwire to Attract Ecoli Cells from a Liquid Solution by the Change in Resistance". I'll have to look up some previous experiments related with proteins and hopefully continue on with a project.

Mahalo Nui,
-Tiana

Hi Tiana,

Unfortunately, although these are all interesting projects, none of them involve analysis of proteins.

Are there any projects that involve the use of antibodies? You could do an excellent project if you had a sample of serum or plasma (any species); you could purify the antibodies from the sample and verify your results with electrophoresis.

Is anyone working with recombinant proteins? If you had could obtain a sample, you could grow the host cells (usually E. coli) and purify the expressed protein and verify results with electrophoresis.

Another possibility. Are there any local crops that are important for the local economy? Perhaps you could purify an enzyme or other protein that would be of commercial or therapeutic value and analyze results by electrophoresis. .

You only need one protein to study, so hopefully these suggestions will help you think of something of local interest.


Donna Hardy

Ms. Donna,

No there aren't any projects that involve electrophoresis that I could continue on from.. However the local farm produce idea is a good one. I'll talk more about it with my teacher.

If I have any questions, I'll come back to this and ask..

Thanks for all your help!

Mahalo Nui,
-Tiana