Lysis Buffer Question

[SciB]

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

[kdong73]

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!

[SciB]

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

[kdong73]

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!

[SciB]

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

[kdong73]

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!

[SciB]

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

[kdong73]

Hi,

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

Thank you

[SciB]

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.

[kdong73]

Hi,

Thank you. This is all super helpful.

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

Thank you.

[kdong73]

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?

[kdong73]

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?

[SciB]

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

[kdong73]

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

[kdong73]

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