Identify crosslinked protein with mass spectrometry ? - (Jul/18/2008 )
Hello everyone,
I'm a Ph.D student and i'm working on the identification of proteins that bind to particular dsRNA motifs.
In order to identify them, i've carried out a RNA affinity purification, which consists in an in-vitro transcription of the RNA, its biotinylation and fixation to streptavidin-agarose beads. Then, i incubate the RNA with nuclear extracts, do some washings to eliminate non specific proteins, then I eluate the proteins that are bound to the RNA. After migration on SDS-PAGE, and identification of several bands by mass spectrometry, i have identified several factors (most of them dsRNA binding proteins) but not of them are actually colocalizing with the structure in vivo (which is duable by FISH and GFP-protein).
Following these results, i've been asking myself if our conditions are not stringent enough to only select (or as much as possible) the proteins that bind with a very high affinity to these motifs. I've tried washings with different salt concentrations, and it doesn't really help.
So i was thinking, would it be possible to use the crosslinking (UV or formaldehyde) to be more stringent ?
The ideal experiment would be :
In vitro transcription of RNA, biotinylation (but not fixation yet), Incubation with nuclear extracts, fixation on beads. And then, we could do washings with very high salt concentrations (like 1M, as the covalent bound between RNA and proteins should resists). Then eluate migrate on SDS-PAGE and identify the bands.
My problem is... Do you think it's possible ? I don't know the efficiency of crosslinking but do you think I could manage to visualise the bands with silver staining ?
If you have any suggestions, i would be very thankful !!
Thank you.
FX
I'm a Ph.D student and i'm working on the identification of proteins that bind to particular dsRNA motifs.
In order to identify them, i've carried out a RNA affinity purification, which consists in an in-vitro transcription of the RNA, its biotinylation and fixation to streptavidin-agarose beads. Then, i incubate the RNA with nuclear extracts, do some washings to eliminate non specific proteins, then I eluate the proteins that are bound to the RNA. After migration on SDS-PAGE, and identification of several bands by mass spectrometry, i have identified several factors (most of them dsRNA binding proteins) but not of them are actually colocalizing with the structure in vivo (which is duable by FISH and GFP-protein).
Following these results, i've been asking myself if our conditions are not stringent enough to only select (or as much as possible) the proteins that bind with a very high affinity to these motifs. I've tried washings with different salt concentrations, and it doesn't really help.
So i was thinking, would it be possible to use the crosslinking (UV or formaldehyde) to be more stringent ?
The ideal experiment would be :
In vitro transcription of RNA, biotinylation (but not fixation yet), Incubation with nuclear extracts, fixation on beads. And then, we could do washings with very high salt concentrations (like 1M, as the covalent bound between RNA and proteins should resists). Then eluate migrate on SDS-PAGE and identify the bands.
My problem is... Do you think it's possible ? I don't know the efficiency of crosslinking but do you think I could manage to visualise the bands with silver staining ?
If you have any suggestions, i would be very thankful !!
Thank you.
FX
You may want to try blocking with BSA or tRNA to prevent non-specific binding. You might also be able to try some detergents to help with the stringency.