SDS PAGE buffers cost saving
david.logan at plant-sciences.oxford.ac.uk
Wed Mar 13 04:42:58 EST 1996
>>An unrelated comment on SDS-PAGE: We are constantly out to save money.
Since the lower
>>electrode buffer is simply for electrical contact with the gel, we use a
cheaper buffer in it,
>Simply make up Tris pH 8.8 at about 50 mM, leave out the glycine and SDS.
Saves a few dollars
>>and is much easier to make up gallons of the stuff (no foaming etc).
>In the Hoefer and Bio-Rad units, the lower buffer is usually several times
>larger in volume than in the upper buffer. Hoefer suggested that you reuse
>the lower buffer several times, but replace the upper. Some users have
>also reduced the concentration--note that this affects the conductivity, so
>if you use this, use it all the time for consistent runs.
>We have tried Walt's idea many years ago and can suggest from experience DO
>NOT REUSE the lower
>electrode buffer unless your life depends on it! The pH of the buffer
>drops precipitously after
>only a single use. This has quite an effect on protein migration in the
>bottom of the gel
>(which now becomes relatively acidic, the bromophenol blue turns yellow
>after 2 or 3 reuses!)
>and gives very inconsistent runs. We find we can get away with a single
>reuse, but it is too
>risky to consider any more.
>An interesting side-effect of my original suggestion (to simply use tris in
>the lower electrode
>buffer) is that when long gels are partly destained, there is a large frown
>evident on the
>bottom of the gel. We presume this is leaching of the SDS from the bottom
>of the gel during the
>16 hrs or so we take to do our runs. We have found it has no consequence
>on protein resolution
>after complete destaining.
I would have to disagree with part of the above. If you are using large
tanks such as the Biorad protein I or II systems (I is the same as the
Hoeffer system) then you can happily reuse the lower buffer (not the upper
one) many times (I tended to stop at three total i.e. two reuses). I have
done this for 4 or 5 years without any problems. No apparent change in pH
buffering, the bromophenol did not change colour. No effect on the migration
quality, simply slightly slower migration (as I do overnight runs there was
no problem - its only slightly slower). Why Phil's buffer is causing the
bromophenol to change I don't know but is probably due to over-reusing the
buffer - its not an infinite thing. As for just using tris in the buffer
well, I hope you don't quote the Laemlli reference when you publish :-).
Personally I wouldn't change it. I'm not surprised there is a large frown on
the bottom of the gel. I've seen worse when I once forgot to add SDS to the
buffer - the run was awful, really bad resolution (wide fuzy bands).
David C. Logan
* david.logan at plants.ox.ac.uk *
* Department of Plant Sciences *
* University of Oxford *
* South Parks Road *
* Oxford *
* OX1 3RB *
* Tel.(direct): (01865) 275024 *
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