Denaturing Proteins

[Hphan30]

I want to make a science fair project on testing how pglo reacts to being put under heat and freezing including being baked into a cake. How can I test this to find out if the protein denatures or not and what other proteins would be easier to test with?

[VSegarra]

Hi Hphan,
Protein stability if a fascinating topic! Could you be more specific about the project you would like to perform in the context of "pglo"? The term you used-- "pglo"-- reminds me of pGLO, a plasmid DNA from BioRad that is commonly used for teaching purposes. Looking forward to hearing back from you.
Veronica

[Hphan30]

Thank you for responding Veronica,
I would like to grow and extract the protein, GFP that is manufactured by pGLO, from the bacteria E. Coli to start off (transforming the bacteria using extracted GLP from the jelly fish Aequorea victoria). I have experience with doing this but after this process and would like to experiment on how stable the protein is when it is put under different ranges of temperatures, the lowest being in liquid nitrogen and I am still researching on what available equipment I can access for the highest temperature. To test this out, I plan to leave GFP in the set temperature to 12 hours and then compare the protein to what it looked like just after I had extracted it from the E. Coli. I think that since GPF will glow a green color when put under a black light, this will be easier for me to check whether or not the protein has denatured or been damaged enough so that it cannot glow. I will also compare intensity of the glow with pictures.
Would this experiment be suitable?

I am still looking for alternative proteins I can test that would provide better results and still deciding if I should test other proteins along with GPF.
This would be helpful to those who want to work with proteins under different temperatures and where the limit of the proteins lie before it denatures or becomes ruined.

[VSegarra]

Hi Han!
Sorry for the delay in responding to you. I think in principle the experiments you are interested in doing would work. In fact, there are a series of kits sold by BIO-RAD that would make it easier to gather most the materials you need for these experiments. The kits also provide you with protocols to follow for introducing pGLO into bacteria (bacterial transformation), growing the bacteria, and purifying the protein from the bacteria using mini-columns. I tried to find a BIO-RAD link that would allow you to place an order but was not successful. I came across a website that describes nicely the system, although it is not a BIO-RAD website (faculty.sdmiramar.edu/dtrubovitz/micro/pglo/). From the way you described what you want to do it seems you already have access to one of these kits. Is this the case?
While the kits provide you with many of the materials to carry out these experiments, you would still need access to some other materials such as: flasks and an incubator to grow the bacteria, as well as a mini-centrifuge. Do you have access to these?
I also have an idea to expand on your project. You can test what effects different reagents have on the stability of the protein when subjected to different temperatures. For example, glycerol is commonly used as a cryo(cold)-protectant for proteins in solution. It would be interesting to see if similar compounds of interest have the same effect. What do you think?
Veronica

[JohnnyNgo123]

Hi Veronica! I'm Johnny, Han's partner in this project.

We have many resources avalible for this project because our instructor has these equipments in our classroom avalible for student usage. We don't necessarily have the kits from BIO-RAD, as mentioned, but we will be using the equipments provided by our school. We have access to a spectrophotometer, centrifuge, and the materials necessary for chromatography.

I am interested in your ideas for expansion! Where can I find other examples of reagents on the internet or is it possible if you can supply us with more?

Thanks for the help!

[VSegarra]

Hi Johnny!
Glad your project is moving forward, great! Some other examples of cryoprotectants widely used when exposing proteins to very low temperatures include:

Ethylene Glycol
Polyethylene Glycol
Paraffin Oil
Ethanol
Sucrose
Glucose

(see hamptonresearch.com/menus.aspx?id=3&sid=189 for more information)

You can select a few of these (whichever ones are easier to find) and compare them. For each one you could even determine what would be the best concentration of the compound to use at a variety of temperatures.
What do you think?
Veronica

[donnahardy2]

Hi Johnny and Han,

It sounds like you are working on a really great project! Veronica has given you a lot of good advice that should be helpful for studying protein denaturation. I have just one additional suggestion. Since growing and purifying green fluorescent protein will takes lots of work, it might be easier and a lot less expensive to do this project using egg white, which is composed almost entirely of a single protein, albumen.

This protein solidifies when it is denatured, so you would need a method for measuring this transformation. And, it would be more difficult to detect the protein in the cake-baking part of your experiment. Perhaps you could investigate using a method to tag the protein with a fluorescent label to make the protein visible.

http://www.ncbi.nlm.nih.gov/pubmed/21909885

Donna Hardy

[Hphan30]

Hi Donna!

Thank you so much for the website! I think I have a good idea of what I'm going to do for my project now. Unfortunately I don't understand how to use the website to find how to connect GFP to a protein, specifically any egg protein.

[donnahardy2]

Hi Han,

To add p-glo to another protein, you would need access to a molecular biology laboratory. The experiment would involve splicing the DNA of your protein to the DNA of the p-glo DNA and inserting this into a plasmid for protein expression. This is a complicated experiment and I think it might be better to use a protein that is readily available, like egg albumen. If you want to use p-glo, you can express this protein alone using the procedure from the classroom kit that you are familiar with.

The procedure I posted is a method for adding a small fluorescent molecule to a protein and involves a chemical reaction. This is a simpler method to obtain a fluorescent protein compared to transformation and protein expression techniques.

What is the purpose of your project? What research question are you trying to answer. Why do you need a fluorescent protein? Please let us know so we can help with the experimental design.

If you are studying cryoprotectants, I don’t know why you couldn’t use egg white protein. This is a pure protein that is naturally translucent and turns opaque when denatured. You should be able to observe the denaturation of the protein using a light defraction technique.

Donna Hardy

[Hphan30]

I've been searching on where to order GFP, unfortunately, I've only been able to find antibodies for GFP and not the purified protein. Are thre any companies that you recomend to us so that we may order the protein?

[donnahardy2]

Hi Han,

GFP is available to order from a number of companies. Try searching for “GFP source.” Here is an example of one company that sells a number of different fluorescent proteins, including GFP.

http://products.creative-biolabs.com/sy ... ch_GFP.htm

The pricing is not available on-line, but I suspect that the cost will be prohibitive for use with a science fair project. Proteins like this are usually sold in milligram quantities, which is not a lot of protein.

Why don’t you tell me more about the purpose of your project and your ideas for an experimental protocol? I think that you need to carefully consider your choice of proteins and select something that is inexpensive and readily available, or work on very small scale. I do not understand why you need a fluorescent protein for your experiment, so please let me know what you are thinking about doing.

Here is an example of a research paper on heat shock of proteins. The entire article, including the materials and methods section is available for you to read. The authors used beta-galactosidase.

http://www.jbc.org/content/266/21/13941.short

Here is another example of a paper investigation the kinetics of aggregation of proteins exposed to heat.

http://pubs.acs.org/doi/full/10.1021/ma9905775

And, here is a similar paper investigation the effects of pH and temperature on GFP:

http://www.springerlink.com/content/176 ... lltext.pdf

Search for “Google Scholar,” and in this site, do additional searches on your topic by searching for “heat denaturation of proteins,” to find similar papers.

Donna Hardy

[HeatherL]

Hi Han,

Bio-Rad (http://explorer.bio-rad.com) sells kits for transforming bacteria with GFP and for purifying proteins from transformed bacteria. They are expensive, though.

I used to work with an outreach group in San Diego called ScienceBridge, and they used GFP in their activities. Try going to the website for their technology sites (https://sites.google.com/site/sciencebr ... sites/home), and contact the Castle Park High teacher listed there. (We cannot give names and contact information on the Ask an Expert forum, but you will find the contact info you need on the website.) Tell her that you are a high school student trying to do a science fair project, and you were wondering whether you could get some purified GFP for your project. You may need to pay for shipping (depending on your location), but if you send a nicely worded e-mail, I think she can help you.

Good luck, and keep us posted on your project!

Heather