ribonucleotides TLC - (Jun/07/2013 )

I use Thin layer chromatograph so often in my research using radioisotp labeled mono ribonuctiodes. But have not seen this before. I had three samples that devolved in TLC and when I mixed them in equal radio one of the spots faded out. Knowing that all samples are in the same buffer. What could be the reason? Please see the figure that may help to understand the problem.

Could be that there is some binding between two or more of the samples - i.e. formation of ds RNA. Try looking at a shorter exposure to see if there is a change in intensity of some of your very saturated image.

Thank you for reply. But this is very unlikly to happend because this is mono nucleotides from same base but differ in number of methy groups. I already tried short expousre and the results pretty much the same.Very confusing!

you mixed equal amounts of each but are they equal to the individual spots?

you'll notice that the shape of spot 1 and the size of spot 3 are altered, as well. the spots in the mixture are affecting the migration of each other. try the combinations of 2 at a time (1+2, 2+3, 1+3) and compare the effects.

Hi medfenko,
I loaded in the mixture alomost 1/3 of the amount of individual spots.

so, then the spots should be lighter and/or tighter.

try loading the individual samples with the same amount of each in the mixture.

I will but what if the effect caused by unlikely but mesterious interaction between two nucleotides. While the main purpose of this experiment is to use them as a standerd mixture!

it's unlikely to be a chemical interaction. it's most likely a physical interaction.

you can also try reducing the amount of each component in the mixture.