fluorescein glycine amide: labelling

[tatituriraju at gmail.com]

Dr Engelbert Buxbaum wrote:
> Raju Tatituri wrote:
>
>
> > I am working with a hyperglycosylated lipid molecule and the MALDI mass is
> > 25000-30000 and it looks like a big smear on the SDS-PAGE gel. But after
> > labelling I can see the molecule as single protein band. I followed your
> > protocol. I am unable to interpret my results.
>
> Carbohydrate moieties in blycoproteins often contain acidic groups
> (carboxylic acid, sulfonic acid), and since the sugar side chains are
> not homogeneous the number of negative charges varies and hence
> migration in SDS-PAGE. This results in the broad bands you observed.
> Presumably some such carboxylic acids were modified by the reagent,
> reducing charge inhomogenity and resulting in crisper bands.
>
> Another way to get crisp bands with glycoproteins is to use the
> positively charged detergent CTAB instead of the negatively charged SDS
> (Anal. Biochem. 314 (2003) 70-76).#

Dear Dr. Engelbert Buxbaum,

Thanks for your e-mail. I did not know how to check the reply from you
but luckily when I typed my name in google I found it!!.

As you know now, after labelling with fluorescein glycine amide we were
expecting to see a broad band as we normally do on a SDS-PAGE gel
rather than a thin and crispy one as we see now. I normally use silver
staining to visualize the molecules on the gel (I use 15% acrylamide).

The subject under investigation is not a glycoprotein but a
hyperglycosylated lipid. We are only speculating that it might contain
a uronic acid residue somewhere in its structure. We don't where it
might be too.

Now after labelling, we see this thin band (like a line drawn with a
pencil) at the position where we normally find a big smear. I forgot to
inform that we also observe a smear-like/broad band at the bottom of
the gel. We don't know what this is.

Conditions of the experiment:

I am performing the experiment in the dark.
I sometimes observe that fluorescein glycine amide is not well
dissolved in water and hence I add phosphate buffer pH 7.4 thinking
that it will help but I still see there is a lot of undissolved
substance (orangish in colour) floating on the top of the solution as
well as sticking to the container. But the dissolved substance is
fluorescent green. After incubating in the dark I dialyse (also in the
dark) in water.

Amount of material: 500 micrograms.
Dialysis was performed in a 1ml eppendorf. The total sample volume is
500-750 microlitres.

My explanation for this narrow band on the gel: Since we are
speculating the presence of a single uronic acid moiety in such a big
molecule it is of no surprise to see one narrow band. Do you agree with
me??

I would very much like to read your comments which will be very
precious to me.

Thanks once again for your time and guidance.

Yours sincerely,
Raju