Deitiker, Philip R
(by pdeitik from bcm.edu)
Tue Jan 10 12:14:20 EST 2012
I think for most enzymes they give optimal conditions, so you make the buffer they say to use or use the one they supply, or as is the case order the buffer from NEB or the enzyme supplier. The level of K+ or Na+ in most cases is not going to make that much difference. For some proteins, like globins, its best to dissolve them in cold water and then dilute them in the working buffer (usually PBS).
On the issue of pH, if you are using extremely dilute buffers, anything can make a difference, and deionized water is not a clearly defined reagent. Deionized water is produced from RO membranes, the membranes age over time, many institutions add salts to the water to protect the pipes, etc. One common thing that is added in areas where the water is acidic is calcium salts to protect the copper pipes from pitting. Raising the pH past neutral also protects the pipes (calcium and copper interact, and higher pH lowers the rate of oxidation). But raising the pH converts acidic ammonia into dissolved ammonia gas, which has greater mobility. Ammonia is not technically added to water but ammonia adducts are added when the level of chlorine is too high to add more. I don't know any lab, and I have worked in quite a few over the years, that uses RO deionized water directly. IOW it cannot be counted on, therefore go for the ~18 mega-ohm water, purchased water, or double distilled. You would be better off purchasing distilled water from the grocery store than using 'house' deionized water. It is clearly true that more most biochemistry you don't need 18 mega-ohm water as a buffer starting mat, but you do need something more reliable than deionized water.
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of DK
Sent: Monday, January 09, 2012 2:08 AM
To: methods from magpie.bio.indiana.edu
Subject: RE: 10X PBS
In article <MPG.29740567a625f6369896b0 from News.Individual.DE>, Dr Engelbert Buxbaum <engelbert_buxbaum from hotmail.com> wrote:
>Btw, if one wants to work with intracellular enzymes, PBS should not be
>used, and replaced with a high K, low Na medium!
Well, if we are that strict then chloride salts should not be used either :-)
Intracellular Cl- concentration is pretty low and most free anions are
large organics. For "pseudo-intracellular" solution, I've isethionate in
one system and glutamate in another. Some people like using tartrate.
To be sure, it does make a difference. Halide ions bind proteins a lot.
P.S. Recently I came across your book, Engelbert. Pretty good!
Made me envious :-)
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