smeared silver stainning on acidic native page - how can I sovle smeared silver staining? (Jun/17/2009 )
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
this may be caused by the buffer that the sample is in (prior to addition of 5x sample buffer).
mdfenko on Jun 17 2009, 01:02 PM said:
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.
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.