Labeling Chemistry Support—Troubleshooting
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Not necessarily. A low level of fluorescence output may be observed when too much dye is attached to the molecule, effecting dye-dye quenching. Also, if the fluorophore is conjugated on or near a micro-domain that is not optimal for the fluorescence output of the dye, this may also result in low fluorescence. This is more pronounced for environmentally-sensitive dyes but may occur for non-environmentally sensitive dyes as well. For example, conjugation too close to aromatic amino acids or a cluster of aromatic amino acids or too close to some bases may result in quenching.
A degree of labeling (DOL) determination would help you assess the level of dye actually attached to the molecule and help resolve what may be causing low signal.
Other methods of post-reaction purification include affinity resin chromatography, dialysis, filter-based spin columns, HPLC, TLC, capillary electrophoresis, and PAGE. Whatever method was typically used to purify your molecule may possibly also be used to purify the labeled molecule as well, considering the difference in molecular weight or charge that the label may contribute to the final conjugated molecule.
Most likely there were lysines on or near the antigen binding site on the antibody. The presence of the bulky labels and/or modifying lysines with another molecule altered the ability of the antibody to recognize its epitope. The SiteClick™ labeling kits allow you to attach label only to the Fc region of whole IgG, leaving the antigen binding site unmodified.
You are modifying the properties of your molecule— a lysine group is capped with a large bulky dye, a carboxyl is being capped with a biotin, or a thiol is labeled with a hydrophobic linker— you are exchanging the contribution of one group (amine, thiol, carboxyl, etc.) for the properties of the label attached thereon.
Most often this occurs when too much label is attached. Lower the molar ratio of label to molecule to limit the amount of label attached to your molecule.
For Research Use Only. Not for use in diagnostic procedures.