fluorescein glycine amide: labelling

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Sun Sep 3 22:09:58 EST 2006


tatituriraju at gmail.com wrote:



> 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.

It usually helps to dissolve the label in a water-miscible organic
solvent like DMSO, DMF, alcohol or the like as concentrated (100 to 1000
times) stock solution and to dilute this into the labeling reaction.
Check the solubility and reactivity of your label in solvents. In
extreme cases the presence of a non-reactive detergent can help. 

> 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??

If you have a defined number of acidic sugars in the carbohydrate the
band will be narrow no matter what the nuber is. Broad bands result from
inhomogenity, that is a varible number of charged groups, as is typical
for glycoproteins. If the labeling reaction converts the majority of the
carged groups into uncharged ones, inhomogenity is reduced and bands
become sharper.


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