I am having trouble with what seems like it should be a simple technique;
my problem is that I don't have anyone around me who does it, so I am
trying to collect advice at a distance.
I want to separate neutral glycolipids extracted from rat brain on TLC
and then immunostain them with an antibody to a carbohydrate moiety.
I have been doing the following:
1. apply the extract, in 50/50 chloroform/meOH
2. run the plate in the chamber
3. fix in polyisobutylmethacrylate in either acetone or hexane (dissolves
much better in acetone)
4. treat the plate much like a sheet of nitrocellulose after SDS-PAGE for
a western; that is, block with BSA, incubate in primary, wash, incubate
in secondary, wash, develop with DAB.
In between the steps involving organic solvents, I dry the plate with a
hairdryer.
I have had the following problems:
1. Silica gel coming off the plates in hunks. I use Aluminum-backed
HPTLC plates, these are better than glass-backed, but I still lose it.
1a. "bubbles" in the silica gel upon spraying the plate with the blocking
solution. Perhaps due to inadequate "fixation" with methacrylate? Or
something else?
2. High background. Too much washing and long incubation times cause
problem #1 to get worse; yet the background on these plates is very high
compared with a Western.
3. Low (or no) signal. I am using a positive control (meconium) that has
the glycolipids I'm looking for in brain. I am loading 10 ul of 1 mg/ml
glycolipids on the plate. In 5 tries, I have only gotten a signal once,
for sure, and once if I squinted and used my imagination.
Does anyone out there have any positive experience with this technique?
How much glycolipid can you expect to see by this method, is it more or
less sensitive than a Western?
How do you load the extract on the plate without it spreading too much?
Will a Hamilton or other small-bore syringe be useful?
Is there some "trick" to the methacrylate fixation that I'm not getting,
other than dip the plate in for 90 seconds to a couple minutes (until "wet")?
Are there some secondary antibodies that are known to be especially good
for this application (i.e. yield low background)?
Does it matter what kind of BSA you use for the block (cheap vs. expensive)?
Would detergent help or is that going to be a disaster for the lipids and
the silica gel?
Thank you for your time and consideration,
Sincerely,
Karen Allendoerfer (a clueless neuroscientist)
