14C-galactose

Separation of radioactively labeled substrate and product on diethylaminoethyl (DEAE) cellulose 14C-galactose is eluted by H20, 14C-galactose-l-phosphate is retained 14C-galactose-l-phosphate is subsequently eluted by 100 mM HC1. Determination of eluted radioactivity in a liquid scintillation counter (/1-counter) [5,... 

Every new charge of DEAE cellulose ( form) should be tested. Dry 10 d each of 14C-galactose and 14C-galactose-l-phosphate with nitrogen and dissolve in 300 pi H20, each. Put 100 pi of each into a counter vial containing 15 ml of OptiPhase (= 100%), 100 pi of each onto columns with 1 ml DEAE cellulose, and 100 pi of both onto a third column (mix). 

Elution with H20 five times 2 ml, collect in five counter tubes containing 15 ml scintillation liquid. 14C-galactose is eluted. 

Separation apply 100 pi from the assay and blank supernatants onto one column each. Wash the columns five times with 2 ml of H20 (discard the eluate, containing 14C-galactose). Elute 14C-galactose-l-phosphate with 2.5 ml of 100 mM HC1 into a counter vial containing 15 ml of scintillation liquid. Repeat the elution with... 

Sample value (dpm) total counts of the two vials of 14C-galactose-l-phosphate elution. 

Separation of the radioactively labeled product from the labeled substrate on a DEAE cellulose column with 20 mM HC1,14C-galactose-l-phosphate is eluted and UDP-14C-galactose is retained. The latter is eluted with 100 mM HC1. Radioactivity is determined with the aid of a scintillation counter (/ -counter) [unpublished]. 

I. Galactose-1-phosphate, 28.8 mM galactose-1-phosphate dipotassium salt (FW variable, calculate the concentration for each batch). Dissolve in H20. 14C-Galactose-l-phosphate 14C-galactose-l-phosphate disodium salt. Dry 20 pCi of the solution under nitrogen and dissolve in 1 ml H20. 

II. Purity test of 14C-galactose-l-phosphate dry 10 pi of 14C-galactose-l-phosphate with nitrogen, dissolve in 200 pi of H20. Put 100 pi in a counter vial with 15 ml scintillation liquid (= 100%) and apply 100 pi on a DEAE column. Elute stepwise as described in a) and b) above. Contamination, eluted with 100 mM HC1, should not exceed 0.1-0.2% of the total radioactivity eluted. 

In brief [43, 44], primary neurons were prepared from the cerebellum of 6-day-old mice and the cells were treated with different concentrations of the target compounds for 24 h. Radiolabeled 3-[ 14C] serine or [14C]galactose was added to the culture medium and incorporation into newly synthesized sphingolipids was analyzed after labeling for 24 h. Lipids were extracted, separated by thin-layer chromatography, and visualized with a phosphoimager. Radioactivity found in the selected lipids is expressed in relation to untreated cells. 

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