Need Help on TLC to separate nucleotides

M. Johan Broekman hanbroekman at csi.com
Tue Jun 20 21:18:22 EST 2000


Hui Wang wrote:
> 
> Hi
>    I am trying to sparate AMP, ADP, ATP using TLC PEI-cellulose plate. I
> do not know how much I need to spot on the plate and what the development
> solution(s). I tried once using 5 ul 100uM ADP/ATP, then developed in 1M
> acetic acid twice and once in 1.2M LiCl (one method found in a paper), but
> I did not see any signal under UV254. If you have that experiences, ccould
> you please share with me at wang.508 at osu.edu?
> Thanks a lot

Do you see anything when using UV254 *before* developing the TLC plate? 
If not, you may be using the wrong plate.  You need to have plates
impregnated with fluorescent indicator, so the spots show up dark on a
fluorescent background.

We are using a different TLC system for separating nucleotides. 
Advantage better separation of metabolites.  Disadvantages: smelly, 5-6
hr development time.  See:

J. H. F. Drosopoulos, M. J. Broekman, N. Islam, C. R. Maliszewski, R. B.
Gayle, III, and A. J. Marcus. Site-directed mutagenesis of human
endothelial cell ecto-ADPase/soluble CD39.  Requirement of glutamate174
and serine218 for enzyme activity and inhibition of platelet
recruitment. Biochemistry:accepted, 2000.

R. B. Gayle, III, C. R. Maliszewski, S. D. Gimpel, M. A. Schoenborn, R.
G. Caspary, C. Richards, K. Brasel, V Price, J. H. F. Drosopoulos, N.
Islam, T. N. Alyonycheva, M. J. Broekman, and A. J. Marcus. Inhibition
of platelet function by recombinant soluble ecto-ADPase/CD39.
J.Clin.Invest. 101 (9):1851-1859, 1998.

A. J. Marcus, L. B. Safier, K. A. Hajjar, H. L. Ullman, N. Islam, M. J.
Broekman, and A. M. Eiroa. Inhibition of platelet function by an
aspirin-insensitive endothelial cell ADPase.  Thromboregulation by
endothelial cells. J.Clin.Invest. 88:1690-1696, 1991.

A. J. Marcus, M. J. Broekman, J. H. F. Drosopoulos, N. Islam, T. N.
Alyonycheva, L. B. Safier, K. A. Hajjar, D. N. Posnett, M. A.
Schoenborn, K. A. Schooley, R. B. Gayle, III, and C. R. Maliszewski. The
endothelial cell ecto-ADPase responsible for inhibition of platelet
function is CD39. J.Clin.Invest. 99 (6):1351-1360, 1997.

-- 
Best regards
Han Broekman
(Please answer to the newsgroup)






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