buffer composition - (May/23/2006 )
ok I will do the same. But suggest my about second Question.
I am preparing this buffer for freeze thawing poces for protocol for protein extraction.
But what i am thinking to do. Use this buffer and lysozyme and keep it on 37 degree for one hr to check the expression in soluble form. Is it ok
Has I thought, you need to prepare 1M Tris - HCl solution.
When a protocol says Tris buffer, is always Tris -HCl. Because is a buffer which pH is adjusted with HCl.
I don´t think you will require NaOH to adjust pH but HCl.
Prepare stock solutions in separate: 1M Tris (pH 8.0) and 1M NaCl . You will certainly need them in other ocasions.
Then from this prepare your buffer mixing the right amounts of solutions (check what I said above).
You don't need to recheck pH again.
You should autoclave this buffer if you want to keep it for using when necessary.
Sorry I cannot help you on that.
So far I didn't isolate protein from bacteria so I cannot advise you on that.
I know lysozyme is an enzyme that is quite effective lysing bacterial and yeast cell wall.
I used lysozyme to extract plasmids from yeast cells. And I remember that is very important to dissolve lysozyme at pH 8.0 because that's the pH of optimum activity of the enzyme.
If you need to prepare lysozyme yourself from the powder take this into acount. You can find in Maniatis & Sambrook the information how to prepare lysozyme.