about detect modified nucleotides using TLC
Posted: Thu Jan 24, 2008 1:51 am
Hi
I just started a postdoc job working on plant mRNA and I got quite a lot of basic questions for the chemists here.
Our lab has been using a TLC protocol (Kieth, 1995 Biochimi) to detect (seperate) methyladenosine from adenosine. Briefly, we label them with 32P-r-ATP using PNK and run 2-D TLC (cellulose). The first solvent is isobutyric acid:NH3 (5:3), the 2nd one is isopropanol:HCl:H2O (75:15:15).
I realized that the temperature can affect the 1st dimention separation. The methyladenosine move further away from the adenosine spot in a hot day, which is very good for us. So I tested it by runing the TLC in a 25 degree oven and it indeed gave a better separation. But it is quite difficult to run TLC in the oven all the time since it meant to be used for northern and southern blot.
Is there any other way to mimic the high temp? Like modify the solvent? and why the seperation is different at higher temp?
Is there other method other than TLC to detect methyladenosine?
Is HPLC-MS sensitive enough to detect it (I estimated that 0.01 pmol of RNA could be detected by this TLC method)?
Thanks in advance
I just started a postdoc job working on plant mRNA and I got quite a lot of basic questions for the chemists here.
Our lab has been using a TLC protocol (Kieth, 1995 Biochimi) to detect (seperate) methyladenosine from adenosine. Briefly, we label them with 32P-r-ATP using PNK and run 2-D TLC (cellulose). The first solvent is isobutyric acid:NH3 (5:3), the 2nd one is isopropanol:HCl:H2O (75:15:15).
I realized that the temperature can affect the 1st dimention separation. The methyladenosine move further away from the adenosine spot in a hot day, which is very good for us. So I tested it by runing the TLC in a 25 degree oven and it indeed gave a better separation. But it is quite difficult to run TLC in the oven all the time since it meant to be used for northern and southern blot.
Is there any other way to mimic the high temp? Like modify the solvent? and why the seperation is different at higher temp?
Is there other method other than TLC to detect methyladenosine?
Is HPLC-MS sensitive enough to detect it (I estimated that 0.01 pmol of RNA could be detected by this TLC method)?
Thanks in advance