Comparing Peanut Proteins and Pea Proteins in Allergies

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Comparing Peanut Proteins and Pea Proteins in Allergies

Postby proscience » Mon Jan 02, 2012 6:23 pm

Hi,

I will be doing your science fair project, "Forensic Science: Building Your Own Tool for Identifying DNA". I ordered the kit from APE. I wanted to do something a little different than just using the food dye included in the kit. I thought about food allergies and how some foods are very similar to others in causing allergic reactions, such as peas and peanuts (both from the legume family).

Can gel electrophoresis separate the proteins in peanuts and peas? If not, what can I use that would be interesting to compare DNA samples that would be ethical for science fair? How would I prepare my samples and what other materials would my mom need to buy that aren't included in the kit?

Thanks!
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby blueswim » Mon Jan 02, 2012 9:00 pm

Hi, and welcome to the forums!
Are you talking about this project:http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p028.shtml ?

Your idea sounds very interesting! :D
Protein electrophoresis IS possible, but it's just a little bit different than the typical DNA electrophoresis used in detective work.

I found this procedure for protein electrophoresis of plants: http://www.sycd.co.uk/who_am_i/pdf/explore/studplant.pdf
It doesn't seem like you need much extra besides the kit, except for the Coomassie blue (well, it depends on a kit... was it an electrophoresis kit?).

Hopes this helps!
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby proscience » Tue Jan 03, 2012 9:14 am

Yes, this is the kit I got.

Thanks for the link to protein electrophoresis. How do I get the pea and peanut to carry a negative charge? I read your first reply but I think my mom won't let me work with HCl. It also looks like something I wouldn't be able to do at home.

I read about extracting strawberry DNA using rubbing alcohol. Once I extract the DNA, can I run it through the gel electrophoresis? What kind of stain would I use? Can the extraction method be used on other fruits?

Thanks!
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby blueswim » Tue Jan 03, 2012 5:05 pm

Hi,
It seems that the link to protein electrophoresis is talking about the Blue Native method. According to some articles I read, Coomassie blue provides the charge for the protein.

As for the strawberries, the same procedure would work on other fruits. I found a tutorial that may be useful: http://learn.genetics.utah.edu/content/labs/gel/

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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby VSegarra » Sat Jan 07, 2012 5:59 am

Hello! Thanks for coming to us for help!

"Can gel electrophoresis separate the proteins in peanuts and peas?" Yes, this is possible. In summary, you can use a protocol to extract the protein material from peanuts and peas. You would then load the sample onto a gel and run it very much like the description provided on the Science Buddies project page you cite above. In the context of a common molecular biology lab setting this would be easy and straightforward to do. You would have all the materials you need. Can you make this type of preparations at home? For some of the aspects of the project, yes. I need to think some more about the details so that I can help you further. Why don't we do some research online and see what we find? I will work on this during the weekend. In the meantime, I would like to give you some clues as well as give you a list of the things we need to research or get more info about.

What I know for sure...
1. Can I use agarose gels to separate proteins? Yes. You can use agarose gels to separate big/large proteins.
2. How can I set up agarose gel electrophoresis? You have the answer to this- http://www.sciencebuddies.org/science-f ... p028.shtml
Is this the kit you purchased?

What I need more info on...
3. Can we design a protocol to extract protein from peanuts etc that does not use any hazardous materials that are also easily available commercially to the common citizen?
4. Once we run the gel we need to have a way to stain it to see the "data" or where the proteins are...can we do this using a protocol that does not use any hazardous materials that are also easily available commercially to the common citizen?

If you wanted to do a similar set of experiments but looking at DNA, questions 3 and 4 would still need to be answered. Usually DNA is visualized using dyes that intercalate into the DNA (and considered mutagenic, lead to mutations). To visualize this type of staining one would need UV light as well. These things are not safe when done outside the laboratory setting. For this reason these type of dyes are probably very hard to obtain outside of the context of the research lab. Also, if you were succesfull at doing this with DNA, you would, essentially be isolating really large pieces of DNA, and you would see "blobs" on your gel after visualizing it. I am not sure you could do much in terms of data interpretation with those pieces of data. This would also sort of be the case with detecting/visualizing overall protein context without more refined specificity.

I will start working on questions 3 and 4 ASAP. In the meantime, you can do the same and we can compare notes. Also, you may want to continue thinking about what other things you'd like/could look at in the context of using electrophoresis as a technique.

A potential idea would be- sticking to samples that, intrinsically, have a way to be detected (visualized) and you do not have to find ways to stain for them. Beets have a very strong color to them- they contain natural dyes that can be separated using techniques like chromatography and electrophoresis. This is the same concept as with food coloring. Can you think of another natural product that has a strong color? Chances are that its "dyes" can also be separated into its different components. You could then compare the electrophoresis patterns. Another idea-You could pick two natural products that share a similar color and see if their "dye components" are similar?
Hope this helps!

What do you think?

How does this sound?



Veronica

PS> Some of your other questions-


"If not, what can I use that would be interesting to compare DNA samples that would be ethical for science fair?"
See above, I think I answered it. Let me know if it is not clear.
"How would I prepare my samples and what other materials would my mom need to buy that aren't included in the kit?"
This is one of the things we need to research.
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby VSegarra » Sun Jan 08, 2012 9:01 pm

Hi,
I found a protocol for isolating DNA from strawberries with reagents that are easily obtained (http://biologyjunction.com/strawberrydna.htm). The problem would be detecting the DNA after you have run your gel. I did not find a protocol for you to do this at home safely. The same idea applies to the protein preparation. So I think we might have to start moving your project idea in another direction. What did you think of the beet juice idea I gave you on my last post? Also, if you want to work on DNA, maybe as a starting point we could use strawberry DNA you isolate using the protocol I provided above. Maybe you could expose the isolated DNA to different stresses- like heat or salt- and see what happens to the DNA containing solution. Just some thoughts.
Were you able to find anything interesting from your search?
Veronica
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby amyc » Mon Jan 09, 2012 2:25 pm

Hi Proscience - You've gotten excellent assistance, already, from Veronica and from Blueswim. I know Veronica has some suggestions for how you may want to redirect your project a bit, and I look forward to seeing how the project shapes up.

When I saw your questions earlier, before Veronica replied, I asked our lead staff scientist to also take a look and see if she had advice to offer. When she looked at your thread today, she noted that Veronica has done an excellent job in offering you feedback. She did say that protein electrophoresis to detect a common protein between peanuts & peas is too difficult, technically, for a home setting, but Veronica has suggested two different approaches... both of which offer great avenues for exploration. Also, she noted that Veronica is correct that most of the methods used in biotechnology labs for visualizing DNA are not appropriate or safe for home use. There is, however, a dye product by Carolina Biological that is safe and appropriate for home use to detect DNA in agarose gels: http://www.carolina.com/product/carolin ... a+stain.do

Good luck with your project!

Amy
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby proscience » Wed Mar 28, 2012 5:44 am

Thanks for all your help! I ended up using the forensics kit to do an experiment on detecting adulterated natural food color using gel electrophoresis. I mixed natural food colors with synthetic food colors and used the gel electrophoresis kit to see if the bands of color for both dyes would separate. Anyway, it was a pretty cool project.

Here's my latest problem:

My teacher took points off my board because I didn't specify brand names in my materials list. She also took points off because I listed the 1/2 teaspoon and 1/4 teaspoon in the materials list and she said it should have been in metric units.

I don't understand why she took points off because I used the material list that was provided in the science buddies kit. Besides, I did this experiment at home. How was I supposed to measure 1/4 teaspoon (1.25 g) of baking soda without an analytical balance? Also, I don't think I needed to list the brand of batteries, microwave, etc. because it shouldn't matter. My experiment wasn't about testing which brand is better.

Anyway, here's my question:

Is the materials list from the sciencebuddies forensics kit in the acceptable format for science fair? Was I supposed to convert everything to metric units like 12 inch alligator clips, 5 1/4" styroform tray, 1 ft. of wire? If so, please let me know ASAP.

Thanks!
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Re: Comparing Peanut Proteins and Pea Proteins in Allergies

Postby amyc » Wed Mar 28, 2012 12:37 pm

Hi -- I'm glad you were able to conduct the experiment! I am sorry to hear, however, that you ran into problems when submitting your display board. Every teacher has his or her own guidelines for a science project assignment. Similarly, science fairs may differ on requirements. So if your teacher requires that you provide additional information about materials -- or use certain units of measurement -- that is what you need to do. Hopefully information provided from your teacher about the assignment detailed those expectations and guidelines.

Our materials lists are provided so that students can procure materials independently (item by item) or in a kit (like the one you used). On the materials lists, we do not list brands unless a specific brand is required. This enables students to procure the appropriate material in the brand that is most accessible to them. In the kits, brands are not provided because the brands included in the kits may change over time. For all materials you use outside of a kit, you would want to list your specific brand information showing what you used to perform your experiment. For the kit materials, you might check with your teacher about noting that it was part of the kit you purchased since brand information is not part of the kit listing.

Ultimately, simply pasting in the materials list from the website may not be sufficient to meet your assignment's parameters.

Measurements, too, will be something that teachers may require be treated using one system or another. In some cases, that may mean tailoring the information from our Project Ideas to fit the assignment or fair rules. Again, you would need to go by your teacher's assignment and guidelines.


If you have questions, let us know.
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