... Dear all,

I am not very familiar with conventional techniques in
glycosphingolipid science, therefore pardon these naive questions:

1- what is your experience in coating whatever type of surface with
a purified GSL (yieldwise). I need to perform binding experiments in
order to fish out a ligand for the forssman GSL (could be protein or
other GSL). I have tried various types of 96-well plates. After
several washings I can still detect the forssman molecule with a
monoclonal antibody but the signal (ELISA type of detection) is very
weak. Starting in the microgram range at the time of coating I find
myself with something that is probably in the nanogram range at the
time of detection. Is there a way of increasing the GSL molecular
density (per unit surface of substratum?). What type of material
would you recommand using for high yield coating?

2- PDMP, PPMP and PPPP are widely described as glycosyl ceramide
transferase inhibitors. What is known exactely regarding their
inhibitoty activity on galactosyl ceramide transferase?
Reproducibly, PPPP failed to exhibit any cell/cell binding activity
whatsoever in our particular model where PDMP, PPMP and even
Fumonisin B1 produce a substantial reduction of binding (WITHOUT
APPARANT APOPTOSIS OR CELL DAMAGE except maybe for a notable
reduction in proliferation pace). I have seen that PPPP would be
relevant for Fabry disease treatment as illustrated by its capacity
to reduce Gb3 levels... has anybody ever checked galabiosyl ceramide
levels as well?

I will be waiting eagerly for your input and comments.

Many thanks in advance

Paola