Protein elution problems - (Sep/29/2008 )
Dear all,
I crosslinked my antibody to protein A-agarose with the DMP/ethanolamine method and performed IP. When I tried to elute proteins from the beads with either 100mM glycine pH 2.5 or 6M Urea (2h/37°C), I could not manage to get the proteins off the beads 
When I added Laemmli buffer I managed to get them. Now I'm trying to elute in 6M Urea plus 50mM DTT.
Would any of you know how I can get proteins off the beads without the antibody? I need to do MS afterwards, either by 2D gel plus MALDI or by LC-MALDI.
Thank you so much for all your help! 
-dnafactory-
QUOTE (dnafactory @ Sep 29 2008, 04:22 PM)
Dear all,
I crosslinked my antibody to protein A-agarose with the DMP/ethanolamine method and performed IP. When I tried to elute proteins from the beads with either 100mM glycine pH 2.5 or 6M Urea (2h/37°C), I could not manage to get the proteins off the beads When I added Laemmli buffer I managed to get them. Now I'm trying to elute in 6M Urea plus 50mM DTT.
Would any of you know how I can get proteins off the beads without the antibody? I need to do MS afterwards, either by 2D gel plus MALDI or by LC-MALDI.
Thank you so much for all your help!
I crosslinked my antibody to protein A-agarose with the DMP/ethanolamine method and performed IP. When I tried to elute proteins from the beads with either 100mM glycine pH 2.5 or 6M Urea (2h/37°C), I could not manage to get the proteins off the beads
When I added Laemmli buffer I managed to get them. Now I'm trying to elute in 6M Urea plus 50mM DTT.Would any of you know how I can get proteins off the beads without the antibody? I need to do MS afterwards, either by 2D gel plus MALDI or by LC-MALDI.
Thank you so much for all your help!
Ok, with 100mM Glycine pH2.5 I got something off the beads but most of it got off only with Laemmli. I thought to wash with 1volume of 100mM Glycine and then incubate 10min in 500mM Glycine pH 2.5. Do you have experience in doing this?
Thanks!!
-dnafactory-
if low pH doesn't work well then you can try high pH.
you can use 100mM triethylamine pH 10 (volatile, smells). neutralize the fractions with 1/20-1/10 volume 1M phosphate pH 6.8.
-mdfenko-