Hi.
I already tried applying a primary anti-fucose antibody to maize mucilage (containing fucose) and a secondary anti-rabbit fluorescent (FITC-tagged) antibody to target the primary antibody. However, I could not image the sugar by a fluorescent device.
It seems that the antibodies do not penetrate the substance (mucilage), because the antibodies alone are imaged and show fluorescence, but not the antibodies + the mucilage.
Might you please share your ideas and experience to solve the problem?
Best regards,
Meisam
Can you partially dehydrate your sample to make it more dense and possibly cut it with a vibrotome to get sections?
Cryosection is another option
Thanks Oscar.
Our gelatinous substance is actually onto a membrane (polyamide). So it is not possible to cut the substance. Neither dehydration worked. The problem is that the antibodies do not bind to our target sugar in the substance.
Meisam,
whether the antibody can recognize its traget (fucosyl residues), or not, may depend on the structure the fucosyl reside has been incorporated into. Could you elaborate on the nature of the antigen the antibody has been raised against? Is it free fucose, fucose linked to a carrier protein (how?) or a larger polysaccaride that contains fucose (how is the fucose connected in the sugar chain, i.e. which glycosidic linkages are there?). Then, you have to match that against the structure of the polysaccharide you are targetting. If the glycosidic linkages are not the same as in the antigen that has been used for the antibody production, it's rather unlikely that your antibody will recognize it.
Regarding the permeability, are there other materials (esp. proteins) that you may detect, in order to exclude that the matrix is significantly contributing to the problem?
If the antibody cannot reach the target by diffusion, maybe applying an electrical field (i.e. doing electrophoresis) might do the trick.
Many thanks Wolfgang.
Our antibody binds to fucose residues bound to the N-glycan in alpha 1,3, which exists in our studied mucilage. Mucilage is a large polysaccharide that contains fucose. The fucose is connected to galactose molecules by alpha 1,3. Therefore, the antigen matches the antibody.
Yes, there are other components in our studied mucilage as well. For instance, proteins (7%) and lipids (3%), in addition to polysaccharides (75%).
Meisam Nazari , hi, thanks for the reply. Yes, it seems that the viscous portion of the mucilage might be a significant barrier for antibodies to penetrate. However, antibodies should be able to recognize the target on the surface of the mucilage. So the goal would be to increase the area/volume ratio by fractionation / or cutting the membrane into tiny cubes of 1 or 2 mm3. Imaging should be done with confocal microscopy to be able to have in focus oblique planes where the antibody is giving a good signal.
I wonder if you could do a partial digestion with a protease or any other not so good enzyme to open up surfaces where the antigen-antibody complex can be imaged.
What is the positive control?
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