Dear Dr. Allendoerfer
TLC overlay works... and can be VERY sensitive depending on the antibody
(well below the 1 pmol level). You may want to look at our recent review,
"TLC of Glycosphingolipids" in Methods of Enzymology 230:371-389 (1994)
[esp. pp. 386-9]
Here are some hints in the meantime:
PLATES: We find E. Merck #5547 to be best for aluminum, #5635 for glass.
It DOES make a difference which manufacturer's plates you use. Plates
that are fluorescence impregnated are sturdier, even though you don't need
the fluorescence as a detection technique (it doesn't hurt to have it
there).
PIBM: I think this is your major problem, in terms of the signal/noise
ratio. The idea is to have the PIBM in a solvent in which it is poorly
soluble (hexane) so that it partitions onto the plate in a reasonable
layer. We suggest making a 10% w/v stock in chloroform, then diluting to
less than or equal to 0.1% w/v into rapidly stirring hexane just prior to
use. Dry the plate thoroughly after TLC development. Then prewet for 30
sec in hexane (alone), then move quickly into PIBM in hexane for 30 sec.
Withdraw and allow to dry without heat first, then you can use the dryer.
The details of PIBM coating are very important.. PIBM BOTH increases
signal and decreases background. There is an optimal PIBM concentration
for each application, although 0.01 to 0.1% should be fine for antibody
overlay. Higher concentrations can dampen the signal, while lower
concentrations will increase background AND dampen the signal. I would
try 0.1% and 0.02% on separate plates to start, just to give you an
indication of whether this range is going to be good for you. After PIBM
coating dry thoroughly (we use a 50 degree oven, but a hair dryer at a
distance should be OK).
BLOCKING: Try prewetting the PIBM-treated and dried plate in buffer or
medium without BSA first, then transfer into BSA-containing medium. This
ensures even blocking.
SAMPLE: 10 ul of 1 mg/ml sounds MUCH too high both in volume and chemical
amount. I would try a range, including MUCH lower concentrations. Apply
1-2 ul to the TLC plate with a Hamilton syringe. Overloading causes
streaking and poor resolution. Did you stain a companion plate with a
char reagent to indicate the quality of the TLC? This would be wise.
Again, read our review for more discussion/details.
OTHER QUESTIONS: Avoid detergents.. they are unnecessary and you run the
risk of solubilizing your lipids. Any old BSA should be OK. For secondary
antibody, we use Vectastain's kit... but any suitable secondary should
work fine (I don't think this is your problem).
FINALLY: Using some antibodies (e.g. A2B5, HNK-1) the technique is so
sensitive that some of the immunodetected bands can not be detected by any
other staining or chemical technique. Whether your antibody and antigen
will give great results is not yet clear, but the technique DOES work, and
I imagine WILL work in your case. GOOD LUCK.
Ron Schnaar
