Bands in IEF gel - How can I extract them? (Mar/08/2007 )
Hi everyone
After running the IEF gel, I want to extract the bands and apply them in an SDS-PAGE gel.
Does anyone have a protocol on how to "cut" and digest the bands in the IEF gel? Can they be stained?
I've already tried the IEF in IPG strips in order to do the 2D, but that didn't work out, so now I'm trying to do the IEF first in a gel and then cut the bands.
I would appreciate some advice.
Thank you! 
After running the IEF gel, I want to extract the bands and apply them in an SDS-PAGE gel.
Does anyone have a protocol on how to "cut" and digest the bands in the IEF gel? Can they be stained?
I've already tried the IEF in IPG strips in order to do the 2D, but that didn't work out, so now I'm trying to do the IEF first in a gel and then cut the bands.
I would appreciate some advice.
Thank you!
to me it is not quite clear what is your aim: Do you need a certain polypeptide/protein for mass spectrometry?
2D-GE with IPG or Zoom strips is totally standardized that alternative methods f.i. first dimension in tube gels, would cause more trouble; try to optimize 2D-GE with gel strips, it should work; however, there are some membrane proteins or large proteins which can make trouble
Thank you for your answer.
My aim is to identify the proteins in the mixture - which seem to be quite similar - and using them in MS. IEF gels seem to be working better than the IPG strips, so I was thinking on doing the isoelectric focusing in the gels and then extract the bands, digest and apply them on SDS-PAGE. Is there any protocol for that extraction and digestion?
My aim is to identify the proteins in the mixture - which seem to be quite similar - and using them in MS. IEF gels seem to be working better than the IPG strips, so I was thinking on doing the isoelectric focusing in the gels and then extract the bands, digest and apply them on SDS-PAGE. Is there any protocol for that extraction and digestion?
some years ago I did tryptic petides to distinguish different phsophorylation status of a protein; peptides were separated in a tricine gel; but you need an IEF tube gel and appropriate equipment; it is not performed in slab gels because of high voltage and heat; an alternative is chromatofocussing
try to find older papers searching by the following keywords "trypsin" "phosphopeptides" "isoelectric focussing"