3 replies to this topic

[wannabe]

I ran acidic native page for my basic protein (pI=9.6) and silver stainned it. but I got smeared bands (attached picture). what makes this?

following is my acidic native page protocol:
separating buffer: 1.5M acetate-KOH pH4.3
stacking beuufer: 0.25M acetate-KOH pH6.8
running buffer: beta alanine 0.35M, galcial acetic acid 0.14M, pH4,3
5x sample buffer: 50% glycerol 1.45ml, 0.25M acetate KOH pH6.8 0.5ml, methyl green traces

used silver staining kit from G bioscience

Attached Thumbnails

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[mdfenko]

this may be caused by the buffer that the sample is in (prior to addition of 5x sample buffer).

[wannabe]

mdfenko, on Jun 17 2009, 01:02 PM, said:

this may be caused by the buffer that the sample is in (prior to addition of 5x sample buffer).

do i need to use specific buffer for acidic native page?
actually, my sample is NGF and the buffer is pbs which is compatible for NGF.

[mdfenko]

the salt, from pbs, will cause problems with page. if you can reduce the salt concentration (by dialysis, dilution, omission) it would go a long way towards improving your result.

Edited by mdfenko, 19 June 2009 - 06:37 AM.