Acidic PAGE? - SDS and native PAGE under acidic conditions (Jun/15/2006 )

Can anylone help?

I am looking for a native PAGE protocol at pH 2-5? My peptide is only stable at this range and aggregates in normal protocols.

Can anyone help?

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pH 4.3 gel (stacks at pH5, runs at pH4.3)

8X running gel buffer:
48 ml 1N KOH
17.2 ml Glacial Acetic Acid
4.0 ml TEMED (or add before pouring gel)
dw to 100 ml
pH=4.3


8X stacking gel buffer:
48 ml 1N KOH
2.87 ml Glacial Acetic Acid
0.46 ml TEMED (or add before pouring gel)
dw to 100 ml
pH=6.7


10X electrode buffer:
31.2 gm Beta-Alanine
8.0 ml Glacial Acetic Acid
dw to 1000 ml
pH=5.0

run "+" to "-"

good luck

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thanks heaps, will give it a try

Rosemary

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Hello,

Could anyone verify if it worked? Just wondering b/c I have a protein soluble in acetic acid and need to run it out on a gel too. We don't have a lot of TEMED left, ordering more, and before I use A LOT (more than a normal SDS-PAGE gel requires) of TEMED I would like some confirmation. Thanks!

Also, how long (approximately) for it polymerize? I can sometimes be impatient and am curious what to expect.

Jerome

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that temed is for an 8x stock. you would use a lot less for an individual gel so you can leave it out of the stock and add directly to the gel mix just prior to pouring and polymerization.

it takes whatever time it takes to polymerize (should be less than an hour, i think). but, be patient. you don't want to run an incompletely polymerized gel.

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PAGE tend to polymerize slow at acidic pH. An alternative is to pre-run a ordinary gel with the buffer you wish to use.

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not a good idea in a discontinuous buffer system.

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Yes. But discontinuous buffer system (pH6.8, pH8.8) does not apply to acidic PAGE.

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this acidic page system is pH 6.7 stacking gel buffer, 4.3 running gel buffer and 5.0 electrode buffer.

most definitely a discontinuous buffer system.

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Indeed, it is a discontinous buffer system. I see many acidic PAGE just use one buffer system, HOAC-pH4.0, +/- urea.

This three pH system seems to work the reverse in principle as the conventional diacountinous buffer system.

Does it give you sharp bands with "stacking effect" on your hand, mdfenko?

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