RNA-IP - (Aug/05/2009 )
I'm trying to perform an RNA-IP (immunoprecipitate a protein of interest and extract the mRNA bound to it). Whenever I lyse my cells with my modified RIPA buffer, I get fluffy white precipitate in my lysate that looks like genomic DNA. Could this contain RNA as well? If I pellet the lysate and use the supernatant for the RNA-IP, could I expect to obtain any RNA from my IP-ed samples?
The composition of my buffer is:
50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors
Thanks!
protein2 on Aug 5 2009, 07:42 AM said:
The composition of my buffer is:
50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors
Thanks!
hi,
i have several papers on this, your RIPA buffer will indeed be removing chromatin (and nucleii)
there are published methods on this, its known as RIP or CLIP
J
Thank you for your reply - but I'm concerned about whether the pellet from my lysate that I will discard (which contains chromatin and insoluble nuclear material) also contains the RNA that I need. Although I have read the published protocols on RIP, they never mentioned anything about losing RNA during the lysis process, so I was wondering if this was a common occurrence.
drjcroberts on Aug 5 2009, 11:37 AM said:
i have several papers on this, your RIPA buffer will indeed be removing chromatin (and nucleii)
there are published methods on this, its known as RIP or CLIP
J
protein2 on Aug 5 2009, 04:42 PM said:
The composition of my buffer is:
50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors
Thanks!
Cellular RNAs are much shorter than the genomic DNA and so should not precipititate out in the same way. I have used RIPA for RNA-IPs and have managed to see my RNA of interest by RT-PCR,
P