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Electrophoresis Buffers - Unlimited Lifespan?

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Old 12-19-2005, 12:52 AM
Wolfgang Schechinger
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Default Electrophoresis Buffers - Unlimited Lifespan?



Dear Experts,

I wonder how long one may use buffers in electrophoresis. I believe
it's aquestion of pH - why else would you use buffers? For conducting
electricity only, NaCl should do as well and for cheap.

At the moment, I am using an older type of the Hoefer SDS-PAGE
apparatur and am running tris tricine gels. Upper buffer (cathode) is
approx 500ml, lower buffer (anode) is estimated 2.5 liters, as I need
it for heat exchange, too.

After 3 runs (4 gels in total), pH changed in both buffers for approx
0,4 units, so I wonder how long I may use them until re-using them will
affect my results.

I thought of re-adjusting pH now. (fighting protons with NaOH and
hydroxyl anions with HCl), I am aware that this will increase the
conductivity of the buffers, as I introduce ions, but that would
increase the available voltage drop at the gel, being in favor of the
system, I'd think. Should I better use TrisBase or Glycine
respectively?

But how far may I go? Probably (at least by Murphey's law), this won't
go indefinitely, even if there are no molds and other bugs growing
after a few (weeks?). Might it be sufficient just to add new buffer to
maintain the volume? Or exchange say 1/10 after every run to get the
whole system into some sort of "steady state?"

In contrst, when one is using TrisGlycine systems, would it be
sufficient to mix upper and lower buffer (which are the same *before*
you run a gel and where H+ and OH- generated during the electrophoresis
/ electrolysis (!) should compensate each other after mixing? The same
then should be true for TAE and TBE in DNA electrophoresis.

Please share your experience. Your comments and suggestions are more
than welcome!

I am also looking for a really good paper that will explain the
electrocheistry behind all that stuff - any refs in your forebrain?

Lotsa Thanks and many delicious Xmas cookies *_*!

Wo

---
Wolfgang Schechinger
Endocrine Research Lab
BG & University Hospital "Bergmannsheil"
Bochum
Germany
[Only registered users see links. ]

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  #2  
Old 12-21-2005, 08:30 PM
D.K.
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Default Electrophoresis Buffers - Unlimited Lifespan?

In article <1134953531.476318.186000@g47g2000cwa.googlegroups .com>, "Wolfgang Schechinger" <[Only registered users see links. ]> wrote:

Either don't pH or use TrisBase/Glycine to do so. By the very nature
of discontinuous electrophoresis, you do not want fast ions in the
running buffer (that's why all protocols say don't pH it)


Sounds like mixing should work in theory (with the unpredictable
side effects related to electrolysis). I'd say it is an easy
enough experiment to resolve the question directly.

DK

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  #3  
Old 12-28-2005, 09:59 AM
Dr Engelbert Buxbaum
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Default Electrophoresis Buffers - Unlimited Lifespan?

Wolfgang Schechinger wrote:


That would increase the conductivity of the system, increasing heating.

The problem is not the OH- and H+ ions, which are balanced by other ions
even in the seperate upper and lower tank buffer. The problem is the
destruction of buffer compounds by side reactions during
electrophoresis.



Ornsteins (1962) original paper on discontinuous electrophoresis
explains things pretty well, for developing your own buffer systems you
should consult Jovins (1973) papers on the subject.
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