Lysis Buffer Question

[SciB]

Probably, although Tris-buffered saline is more commonly used for chromatography than phosphate buffers because some phosphate compounds can precipitate.

If your lab has Tris base, you can easily make a pH7.5 buffer by following the online recipe:

http://cshprotocols.cshlp.org/content/2 ... 11830.full
https://en.wikipedia.org/wiki/Tris-buffered_saline
https://www.protocolsonline.com/recipes ... aline-tbs/

If you don't have Tris, don't worry--PBS at pH 7.5 will work ok 99% of the time.

Keep asking questions.

Sybee

[deleted-591615]

Hi,

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

[SciB]

Hello again,

A diameter of 4 cm would be larger than you would need for the size of sample you are running. I would try to find a glass tube of 1 to 2 cm inside diameter by a length of 30 to 50 cm. In other words, you need a column that is more than twice as long as the syringe you have and about one quarter the width. Protein separation occurs as the molecules travel down the length of the column so usually longer is better. A long, narrow column is better than a short, wide one. The ideal thing to use is a long buret because it has a stopcock at one end so you can control the flow of the running buffer.If you can get a long, straight glass or plastic tube you can attach a piece of rubber to the end and clamp it to act as a flow valve. If you do a search, you can find pictures or videos showing what I am talking about

There are all kinds of formulas for calculating the best length-to-diameter ratio for different column matrices, various types of sample and different running buffers. I don't expect you to do all those calculations, but just be aware that they exist. When you take chemistry (assuming that you decide on a science career) you will learn a lot about chromatography as it is still one of the workhorse methods for separating molecules in the lab.

I thought you would be asking me how much Coomassie brilliant blue (CBB) to add to your protein sample to run on the column and I really do not know. My feeling is that it is not especially important. The only time I have used CBB is to stain proteins that have already been separated by electrophoresis through an acrylamide gel. The gel is placed in a tray with a CBB solution and allowed to stain until the protein bands become dark enough to see easily, so it's not a quantitative method. You can find recipes for making CBB solutions online, so just look until you find one that uses reagents that you have access to. All proteins are stained by CBB, but they can be destained and CBB is not toxic to living cells.

Your column separation method will rely on size exclusion of the protein molecule so any excess dye should run through the column along with the running buffer. Just to be sure nothing weird is going to happen, I would set up the DE column using PBS as the liquid phase and when the column is stabilized try adding 1 ml of CBB and after it enters the DE, start the flow of running buffer at about 1 ml per minute and watch what happens to the dye. Be sure to start a stop watch when you add the running buffer so you can time how long it takes the dye to run the length of the column. What you should see when you load stained proteins on the column is the dye moving down the column while the stained proteins form a band at the top and gradually separate as they move down according to size.

I'm sure you will have more questions. I wish I had more definite answers for you, but this is not a method that I have tried and I found no specific instructions for how to do it. A lot of science is this way, however, and actually it is a challenge that is quite exciting to tackle. Routine methods are...well, routine. The best thing is when you get to use your training and experience to come up with a new technique that really works.

Good luck!

Sybee