Science Buddies: "Ask an Expert"

Hi all,
For my project, I want to test the effects of pathogenesis related proteins in plants on yeast growth inhibition, and I want to extract and precipitate the plant proteins. What is a suitable lysis buffer to use for extraction? PR proteins are acid soluble, and I don't want to denature the proteins so I can test them against yeast growth.

I found some past experiments that used acetic acid or citric acid with 2-mercaptoethanol, but I'm worried that they will denature the proteins. However, I need a buffer with a low pH so that the proteins will dissolve (or is it okay to use a buffer with a medium pH if the proteins are only acid soluble?). I'm not sure what to do...

Also, what would be a good way to precipitate these proteins? (without denaturing them)

Is there a better way I could test the effect of PR proteins on yeast growth?

Thanks a lot!

Hi,
You have the makings of a great project, I think, if you can work out the details.

I am not experienced with plant proteins, only animal. Plants have cellulose and cell walls that makes the cells resistant to the lysis buffers I use with animal cells or tissues.

I did not find one single good reference for a method to extract plant proteins, so I think you will have to piece together a method based on what you can find from the literature and from the resources you have. I am assuming that you have a lab to work in or else this project is going to be a non-starter.

The first step would be to decide on the type of plant and which parts of it to use as your source of proteins. Once you have the plant in hand then the next step is to somehow grind it up finely. Scientists often use liquid nitrogen in a mortar with a pestle to do this, but I am guessing that you may not have access to liquid nitrogen.

If you have a large mortar and pestle, you can grind the plant material pretty thoroughly by hand and then mix it with an extraction buffer. Here is where I can't help you yet because I don't know what the extraction buffer would have to contain in order to be effective. We used to use phenol, but this compound is quite dangerous and can cause serious burns to skin and blindness if you get it in your eyes so that is why most scientists don't use it anymore.

I would recommend doing more reading about lysis buffers for extracting plant proteins and check the websites of various companies that make lysis buffers. Hopefully you can find something that you or your institution can purchase and that does not cost overmuch.

The next thing I would recommend is that you learn about gel electrophoresis of proteins. This technique allows you to separate proteins according to size using DC electric current. If you use a whole protein extract, you will be testing all the proteins and you won't have any way of identifying those specific ones that have effects against yeast. By using electrophoresis to separate the proteins into discrete bands that you can visualize by staining, you can actually cut out specific proteins from the gel and test them individually. It will be a lot of work, but this will be the most scientific approach you can use. If you know the size of the anti-yeast proteins then you could choose to look at proteins of that size first and maybe get lucky in finding the active ones. Once you get to this point after developing the protein extraction method, I will help you with the electrophoresis part.

Hope this helps. I'm sure you'll have more questions, so please post again and I will try to get you started on developing an extraction method that will work for you.

Sybee

Hello,

Thank you so much for your response. It was very helpful.

I'm still looking for a lysis buffer to use. If I want to test the function of the proteins, do I need to worry about denaturing or reducing them during extraction? I read some things about renaturing proteins by removing the denaturant, but they also said that the proteins might not fold back correctly into its original structure. Should I look into using a native buffer?

Also, about gel electrophoresis, I think that it would definitely be a good idea to use it. However, I'm working in a high school lab and I'm not sure whether the school has a vertical electrophoresis chamber. I'll ask my advisor if we do, but if we do not, would it still be possible to test the effects of the proteins on yeast, even if I can't identify specific ones? I know that it is a lot less favorable than if I were able to identify the specific proteins, but lack of equipment might not let me...

Thanks!

Hi again!

Glad my answers were helpful. I'll try to keep giving you useful info so you can do the project that you are interested in.

You want to have active proteins to test on the yeast so, yes, the lysis buffer needs to preserve the native structure of the protein. Here's a good [though technical] site on protein electrophoresis from a company that sells research equipment and supplies: https://www.thermofisher.com/us/en/home ... resis.html

SDS-PAGE is a method in which proteins are denatured by a detergent [sodium dodecyl sulfate, SDS] plus a compound that breaks the disulfide bonds in a protein [mercaptoethanol] and boiling for 3 minutes. The resulting proteins can be nicely separated on a gel but they are probably not active any longer.

What you need for separating active proteins is called native gel electrophoresis. The website I listed above explains how native gel electrophoresis works.

A gel electrophoresis set-up is quite easy to make if you are handy with tools. I would seriously recommend that, if you have time, mechanical skills and a few extra dollars for the supplies, that you consider building an electrophoretic chamber. It makes your project 100% better and you will have the satisfaction and experience of making and using a piece of scientific equipment to do an interesting experiment.

Here are some DIY sites for building an electrophoresis chamber:

1. This one is relatively simple and crude but would probably work.
https://www.instructables.com/id/Buildi ... el-Electr/

2. This project is much more detailed and more like a commercial gel electrophoresis apparatus [more $$]
https://learn.genetics.utah.edu/content ... el_box.pdf

3. Here's another relatively cheap and simple project for building an electrophoresis chamber, so you can take your pick
https://hackaday.com/2017/05/11/get-int ... resis-rig/


There is a potential problem with using these gel electrophoresis chambers for separating proteins and that is the substance that the gels are made of--agar or agarose [a purified form of agar]. This technique was originally developed as a way to separate nucleic acids--DNA and RNA using a DC electric current. All nucleic acid molecules are negatively charged because of the phosphate groups so they will naturally migrate towards the positive electrode, the anode.

DNA molecules are a lot larger than proteins so they need to be run on a gel with relatively large pores so that the molecular friction does not slow them down too much. Proteins, on the other hand, have an atomic mass about one-tenth that of DNA and thus should be run on a gel with much smaller pores. That's why you will see protein gels made from a kind of plastic called polyacrylamide: https://www.news-medical.net/life-scien ... PAGE).aspx

I don't recommend using acrylamide because it is expensive and is very toxic until it polymerizes. You can purchase acrylamide gels for about $10usd each but you do need a different type of electrophoresis chamber to run them and this type is difficult to make.

There is another relatively simple way to separate proteins and that is by chromatography. This involves preparing a column of some substance in a glass tube with a valve at the bottom. The protein solution is put on the top of the column and allowed to run through it and drip out the bottom. As the solution drips out, you collect it in tubes and these are called fractions. A reservoir containing a separation or running buffer is placed above the column and constantly keeps liquid running thru it to move the protein fractions along.

Here's a site that describes protein chromatography: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/

The easiest method of protein separation is chromatography. The degree and type of separation depends on the type of column material and its length. The proteins are moved along with the running buffer so are separated according to size and shape. Their activity is unaffected because they are not denatured before putting on the column.

The two major problems with protein separation are choosing the column matrix and choosing the running buffer. I would recommend doing some online reading about protein chromatography and see what you come up with. You are not looking to do a perfect, quantitative separation of every protein in your samples--just a relatively simple fractionation so that you are not putting everything on the yeast and not know what caused an effect, if you see an effect.

If you decide to use chromatography, I can help you in choosing the column material and the running buffer. You need a glass or plastic tube about 15 mm in diameter and maybe 20 cm long. The longer the column the slower it runs and the greater the separation. You also need a case of small tubes, either plastic or glass--the cheapest you can find (they do not need to be sterile or have caps). These are for collecting the fractions from the column which you will do by counting the drops as they fall into each tube--tedious but doable.

OK. I think that's enough information for one post. Read some about chromatography for protein separation, watch some videos and then decide if you want to try it. Fractionation of your protein mixture, even crude fractionation is better than none. Plus you will learn a valuable chemistry technique that you will undoubtedly use later if you continue in science.

Good luck!

Sybee

Hi again,

Thank you so much!

I'm thinking about doing chromatography. What type of chromatography would be best for proteins? I was thinking about gel filtration since it separates by size, but what types of gel particles would be best to use? Or do you think that there's a better way do separate the proteins?

Also, if I use chromatography, do I need to take anything into account when extracting the proteins?

Thanks a lot!

Gel filtration is what I would recommend and there are many different types of column material that you could use. Do you have access to a chemistry lab at school? They should have Sepahadex which is one of the most common common protein separation materials. It is available in different pore sizes, but what you would need would be G50 or G75. Ask a chemistry teacher if they have this kind of column. The columns are reusable [usually] so should not cost anything.

If you can't get Sephadex I would try diatomaceous earth which I think is available in home supply stores. It is a filtering agent. You can look it up in Google and see who carries it. I don't know how well it would work for separating your proteins but I would try it.

Sybee

Thank you so much!! You've really helped me a lot!!

Also, what running buffer would be good to use? What factors should I take into account when choosing one?

You are welcome! Glad my information is helping.

Ah--you had to ask about running buffer. But you did not tell me if you found Sephadex in your school. I will assume that you did not and that you will be using DE for the column matrix.

The short answer is, I don't know for your protein samples and the DE column what would be suitable but I will look it up.

Here's a detailed method I found as part of a patent application for using DE to purify a specific protein. The summary said that the process could also be used on plant extracts: https://patents.google.com/patent/EP1403274A1/en
Don't try to understand all the technical jargon and details because this was written for a complex industrial process. Just try to get the main ideas. It is way too long and complicated.

How will you break up the plant material first and extract the proteins? There are some non-denaturing chemicals that can be used for this but I don't know if you have access to a school laboratory. Grinding the plant material with a mortar and pestle is probably the easiest way for you to carry out the extraction. But after you do this, you will have to separate the debris from the extract. You can do this acceptably by using a funnel in which you place a coffee filter and collect the clear liquid. This is the 'clarification' step mentioned in the patent procedure.

The next step is called 'capture' and that means putting the extract onto the column. When you do this, make sure there is a container under the bottom fitting of the column to catch the liquid. Also, have all your tubes ready to collect fractions.

The one thing I am not sure of is how proteins interact with DE. Mainly what I am concerned with is do the proteins adsorb to the DE particles. You need to understand the difference between adsorption and absorption. If you put your protein extract on the column and the protein molecules pass through it like a sieve then that is absorption. However, if you put the extract onto the column and the proteins STICK to the DE, then that is adsorption and you then need some chemical way to un-stick the molecules in order to get them off the column.

Before you put the protein extract on the column, it is necessary to wash it by running through several volumes of buffer. You want to load the extract onto a column that is already equilibrated with running buffer.

In reading online procedures, I see that DE will bind nucleic acids which is good because that separates them from your proteins so that they do not interfere with your later experiments.

What you are looking for is an interaction of the proteins and the DE through positive and negative charges and what are called hydrogen bonds. I don't know how much chemistry you have been taught and I have to use terminology that you probably won't understand. All I can say is look it up. You can find everything on the web and learn everything from tutorials on Youtube. Here's a good reference on DE chromatography: https://www.researchgate.net/publicatio ... _Biosilica

The other important thing that you need to know about is pH which put simply is how acidic (low pH) or basic (high pH) a solutions is. The reason pH is important for chromatography is that it alters the charge on a protein and thus affects how that protein interacts with the column matrix, DE in this case.

What I am trying to figure out here boils down to two basic things related to the running buffer: salt concentration and pH, each of which can affect the retention times of the protein on the column.

I think I will stop here because I have hit you with a lot of information and you need time to process it.

Let me know whether you have access to a chemistry lab because this will make a big difference in what you will be able to do and what you can't do.

You will be able to use a DE column to separate proteins in a plant extract, but all the details still need to be worked out. That's why I need you to learn as much as possible as quickly as possible so you will be able to understand the chromatography process. It will all make sense eventually!

Sybee

Hi,

Thank you for your reply.
I'm sorry I didn't clarify this earlier. I don't know whether or not my school has Sephadex. I asked but they haven't replied yet. I do have access to a chemistry lab.

But I'll look into both methods until I receive a reply. If my school does happen to have Sephadex, would it be better to use Sephadex or DE?

Thanks a lot!

Good that you have access to a chemistry lab--that helps a lot.

Sephadex is the standard matrix for size-exclusion chromatography of proteins so I would use that (G25 or G50) if you have it. You might want to consider running your plant extract through a small DE column first in order to remove nucleic acids that might interfere with the proteins later.

Here is a procedure for binding DNA to a DE column. There are some steps that you do not need to do because the procedure is for purifying DNA, not proteins: http://alexandrevallet.free.fr/alex/ava ... tm#binding

Note that the binding buffer contains 7M guanidine hydrochloride. You definitely do not want to add that because it will denature your proteins! I would just use the buffer alone--10 mM Tris-HCl + 1 mM EDTA, pH 8.5 (TE buffer). If you do a search for Tris buffers you will find instructions on how to make them. They are one of the most common biological buffers and someone in the chem lab may know how to make TE, assuming that they do have Tris base or Tris-HCl. If you don't have Tris, you can use phosphate-buffered saline (see recipe below).

For the Sephadex column I would use the standard physiological buffer--phosphate-buffered saline, pH 7.4 (PBS). Here's a recipe for how to make it: https://www.aatbio.com/resources/buffer ... 5-8-to-7-4

Good luck.

Sybee

Hi,

Thanks again for the help. Unfortunately, my school doesn't have Sephadex, so I'll have to use DE. I'm sorry for any time this may have cost you.

The "immobilization of proteins" researchgate article said that there will be less adsorption to the silica if there is a high concentration of ions and a lower pH due to interactions with the silica and proteins and less electrostatic attraction. Should I use a more acidic buffer with a higher salt concentration in order to decrease adsorption of the proteins (or do I want more adsorption)? But hydrogen bonds are also relatively weak and the article said that desorption is likely to occur, so maybe a more acidic buffer with a higher salt conc. isn't necessary? Would the TE buffer or the phosphate buffer work?

Also, for binding nucleic acids to the DE, do I have to take the buffer into consideration? Since nucleic acids have a negative phosphate backbone and the gel particles have nonbonding electrons, wouldn't some chemical have to be added in order for them to attract? Is that where the guanidine hydrochloride comes in? But since that would denature my proteins, I don't want to use that, so do I need to use something else? Or would the TE or phosphate buffers work for that?

Also, when extracting the proteins, I don't need to add any chemicals? Will it be sufficient enough to grind them and put them through filter paper?

Sorry, I know this is a handful. My main questions are would the TE or phosphate buffers work for DE chromatography, do I have to account for nucleic acids binding to the gel particles, and do I need to add any chemicals or take any additional steps when grinding the plants to extract proteins?

I really appreciate all of your help!

Thanks a lot!

Hi again,

I did some more looking into commonly used solvents for column chromatography. A lot of sites said that the most common solvent is a mixture of ethyl acetate and hexane, and most are a mixture of a polar and a non polar compound. Why do most mixtures contain both? Does it help with the solubilities of different substances as they travel through the column? Sites also said that sometimes it is sufficient enough to just use one solvent, such as ethyl acetate. When is it appropriate to just use one solvent? Since ethyl acetate is polar, would it be attracted to the polar DE particles, decreasing adsorption of the proteins? Most sources say that the pr proteins I want to investigate are either acidic or basic and are soluble, so would it be necessary to include a non polar compound in the solvent?
Sorry for the sudden mass of questions.

Thanks a lot!

Hi,

Welcome to the complicated world of chromatography!
I can't answer all your questions, because every question depends on a whole bunch of conditions like the type of column material, the chemical make-up of the protein extract that you are loading onto the column and the structure of the proteins that you are looking to separate.

My strategy would be to start with just a simple polar buffer like phosphate-buffered saline and try it. Your chem lab should have mono- and di-basic sodium phosphate and there should be a pH meter that you can learn to use to adjust the pH to 7.4.

There is one big problem we haven't talked about yet and that is how you will detect the proteins as they come off the column. Normally we would do that by passing the column effluent through a spectrophotometer that would measure the absorbance at a specific wavelength that indicates protein. As a protein passes the detector, there would be a spike in the signal, so you know that fraction contains protein. it does not tell you which protein, just that there is protein and the height of the signal indicates how much. Doing lab science is like cooking or woodworking--it is much easier when you have the right tools!

That's great, IF you have a column running through a spec, but I kinda doubt that is the case. So, what is plan B?

Well, there are dyes that will stain proteins a color so that they are visible, and the most commonly used is Coomassie Brilliant Blue: https://www.thermofisher.com/order/cata ... duct/20279
But don't buy it from Fisher unless you want to pay $130 + $20 shipping!!

You can buy a bottle of Coomassie liquid stain from Carolina Biologicals for $10+: https://www.carolina.com/catalog/detail ... kcEALw_wcB

Carolina Bio also sells lab grade DE--500 g for $16: https://www.carolina.com/specialty-chem ... /857570.pr

I think you can mix your protein extract with some Coomassie stain to color the proteins and then load it onto the column. With luck, the unbound stain will wash through the column, leaving the stained proteins visible so that you can watch them as they migrate down the column and come out the bottom as you collect them into tubes. Then, at least you will have a visual cue for where the proteins are even if it does not tell you what they are.

As to all your other questions, I suspect that at least some of these combination polar/nonpolar buffers were for use with a different type of column matrix--not Sepahadex or DE. There are a huge number of chromatographic memthods described online and that's why I said to try the simple method first. Scientists want to isolate a specific protein in relatively pure form, and that is why they have developed all these exotic methods. They work, but they are way beyond what a high school lab can do.

I'll be waiting for your next set of questions.

Sybee

Hi,

Thank you so much! I appreciate all the help.

There's just one thing. My school doesn't have a chromatography column that I can use, and most that I found online are expensive. Are there any places where I can buy a less expensive column or make a diy column with syringes? I found a column chromatography on this website using syringes https://www.sciencebuddies.org/science- ... grape-soda but I'm not sure how well it will work.

Thanks a lot!

You can use a syringe, but in my opinion this gives to short a distance for the proteins to separate well. You can use any glass or plastic tubing as long as it is rigid and you can attach some fitting to the bottom so that you can collect the samples as they drip out.

I would suggest PVC pipe, but you need to have a clear tube so you can see the Coomassie-stained proteins as they migrate down the column. You could try going to a Home Depot type store, or even a Walmart, describe what you need to some of the sales people and maybe someone can come up with an idea. I'm sure there is some type of clear plastic tube there which is used for something else, even for a toy, that you can cannibalize into a chromatography column. Be creative!

Good luck!

Sybee

Hi,

Thank you so much for all your help!

So, when I'm testing whether the proteins have antifungal effects on yeast growth, do you think that a disk diffusion test would be appropriate?
I would soak small circles of filter paper in the protein fractions, then put them on a plate with yeast cells smeared onto them and see if there are any zones of inhibition, and if there are, which fraction it came from and how large it is.

Thanks a lot!

Yes, a Kirby-Bauer test should tell you if some of the protein fractions are toxic to yeast. The only problem i foresee is diffusion-related. Proteins are relatively large molecules compared to antibiotics and drugs and they don't necessarily diffuse very quickly through agar. It depends on their size and their net charge.

Remember that proteins are made up of a long chain of amino acids and each of these components can have side-groups that are positively charged like lysine's amino group, negatively charged like glutamic acid or uncharged like glycine. The net charge on a protein depends on which groups predominate.

The other way you could test the effect of the protein would be on yeast metabolism. You can put yeast suspensions into a bottle with a cap that has a gas fitting. As the yeast produce carbon dioxide, you collect it in a graduated cylinder so you can measure the volume. A protein might inhibit yeast growth by blocking metabolism of sugar and CO2 production. You would have bottles with just yeast and bottles that had yeast plus a protein fraction and you would compare the CO2 output over a specific length of time and at a certain temperature. A significantly lower CO2 output would indicate that the protein fraction inhibited metabolism.

Post again when you need help.

Sybee

Hi,

Thank you so much. I just have one last question.
How would I collect the CO2 in a measurable way? CO2 is soluble in water, so I'm not sure if water displacement would be the best method? Or would the difference be negligible?

Thanks a lot!

You are right that CO2 is soluble in water--but only to a degree. And remember, the water that you use has been exposed to the atmosphere which contains CO2. You can measure CO2 formation by water displacement and ignore the small loss due to solubility.

Good luck!

Sybee

Hi again,

If I use mortar and pestle to ground the leaves, do I need to use a lysis solution before I run them through the column? Or would the cell membrane break down along with the cell wall? If I need to use a lysis solution, would a detergent be necessary (non-ionizing)? Also, would a chelating agent or protease inhibitors be necessary (most pr proteins I want are protease resistant)?

Additionally, if these were necessary, would I have to somehow remove them before running them through the column (microcentrifuge maybe?)?

Sorry for all the sudden questions and thank you for the help!

Hi,
Don't worry about asking questions--that's what we are here for!

1. grinding the leaves in lysis buffer
Yes, I would have the lysis buffer present during the grinding. Breaking up the plant's cell walls and fibrous material takes some strong action. Use a non-ionic detergent like Tween or Triton if you can get it and add some EDTA to inhibit proteases just in case.

2. Common recipe for lysis buffer
50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Triton X-100, and 5mM EDTA.

You should filter out the undissolved plant materials using a coffee filter but you do not need to remove detergent or EDTA before applying the sample to the column.

Keep posting questions. It is better to check if you are not 100% sure because one wrong step can ruin the experiment. Remember that all procedures need to repeated independently so that you can do statistical tests on the results.

Sybee

Hi,

If I make a solution using phosphate buffered saline, what concentration should it be?

Thank you

Phosphate-buffered saline (https://en.wikipedia.org/wiki/Phosphate-buffered_saline) is probably the most common reagent I use in the lab. All you have to do is Google 'phosphate buffered saline recipe' or even just 'recipe for pbs' and you will find it:

https://www.protocolsonline.com/recipes ... aline-pbs/

The only problem you will have is in adjusting the pH to 7.4 unless you have a pH meter. I forget what pH the solution is when you make it but I think it is close to 7.4. If you have accurate pH paper, you could use that to measure the pH. It should be close to 7.4 but does not have to be exactly 7.40. If you DO have a pH meter then you can make your PBS exactly 7.40.

For 1 liter of 1X PBS, prepare as follows:

Start with 800 ml of distilled water:
Add 8 g of NaCl.
Add 0.2 g of KCl.
Add 1.44 g of Na2HPO4.
Add 0.24 g of KH2PO4.
Adjust the pH to 7.4 with HCl.
Add distilled water to a total volume of 1 liter.

Hi,

Thank you. This is all super helpful.

About how long should my chromatography column be for a good separation?

Thank you.

Also, in some videos and procedures, there is a centrifugation step, and the supernatent is discarded because the proteins are in the pellet. I don't plan to centrifuge my samples, but if I pass them through filter paper, will the proteins be dissolved in the solution? Should I be worried about them staying behind with the debris?

Also, about what mass of leaves would I need to have a sufficient extraction (my plants are not as big as I expected them to be because of schedule delays). What is the minimum amount needed?

OK, lots of questions--let me take them one at a time.

1. Length of chromatography column
Usually longer is better, but remember that the diameter of the column is also important because you want all the extract to enter the column matrix in a few millimeters of head space. I forget--what column material are you going to use?

2. Filtering or centrifuging the extract
The proteins are in solution--at least most of them--so you want to filter the debris and just put the filtrate on the column. A coffee filter [washed] or even some cheese cloth will remove the particles adequately.

3. How much leaves should you use?
That's something I don't know. Maybe you could find a paper that describes extraction of certain plant proteins that will tell you how much material they started with. In biochemistry class we used to joke about purifying something to nothing, but it is no joke-- it can happen if you use too little starting material. But, in your case you are not purifying your material through that many steps so I would not worry about ending up with nothing.

Think about what is in the plant cells and how it might affect the chromatography. The cells have lots of proteins, true, but they also contain a whole genome's worth of DNA plus all the different types of RNA. The cell membranes contain a variety of lipids and the cell walls are made up of cellulose. All these components will go on the column and potentially change the protein migration characteristics. Ideally you would separate all the proteins from the non-protein stuff first and put just proteins onto the column, but that would be rather difficult to do in a high school lab.

I hope this helps. Keep asking questions no matter how unimportant they might seem because there might be something you need to do [or NOT do] that will affect your results negatively. Keep reading about protein separations and chromatography and electrophoresis so you stay up to speed on the methods.

Good luck!

Sybee

Hi,

Thank you for your replies.

How small does the diameter of the column have to be? I have syringes that are about 15 cm long, but have a 4 cm diameter, which I think might be too much... I am using diatomaceous earth.

Also, to follow the proteins in the column, you suggested that I add coomassie stain to stain the proteins. How much stain will I need per fraction? Is it likely that the stain might not bind to all the proteins? Is there any way to differentiate stain that is binded to proteins and stain that is not?

Thank you

Also, for the running buffer, would 1x phosphate buffered saline with 150mM NaCl be ok?