Our lab is doing nanovaccine project. To label the antigen Bm86 (75KDa) which is the cattle tick antigen, we used Cy5 tag to conjugate with the proteins and then loaded them to silica nanoparticles(SNPs) . After the labeling, we conducted SDS-PAGE and western blot to confirm the binding between SNPs and Bm86. However, not like the results we got from the unlabeled one, we could not get any band on sds gel but had a band for positive which is the unlabeled Bm86, and for western blot we can not only see bands on the 75KDa position but on the top of wells. Therefore, we assume not all labeled proteins ran through the gel, some of them still left on the top like the situation when we add insufficient loading dye.
The cy5 dye has around 1 kDa of the size and more than 1 cy5 molecules may bind to the protein depending on the reacting groups available on the protein. So it is quite obvious that the protein's molecular weight and its gel mobility properties change after the labeling. The labeling may also effect the binding properties of the protein to the nanoparticles. Being an immunologist, I also have an additional suggestion. Make sure that the cy5 conjugation does not effect the antigenic properties of the protein and I hope you just use this labeled protein only for the tracking purposes.
If you use too much label, the protein solubility can be loss. Even in sample buffer. If the molar ratio is high for Cy5 and Bm85 during labelling process, lower the amount of the dye.
I shall assume that you ar using the Cy5NHS ester to label your protein. If you have chosen the appropriate labelling conditions, the C5 will label the amino terminal and any available lysine side chain amino groups . It is often the case, in water soluble proteins, for the lysine amino groups to be at the surface of the protein and with react readily with the Cy5NHS ester. It is also the case that there can be as many as 1 in 10 of all the amino acids in protein to be lysine. If sufficient Cy5 NHS ester (a molar excess over the lysine) , is used in the reaction then it is possible for all of the lysine to be labelled with the Cy5. For protein of, say 500 amino acids and a molecular weight of approximately 55000 then about 50 lysine may be labelled which will add approximately 570 daltons to each lysine and therefore the potential MWt of the fully labelled protein will be 835000. This will have major effect on the electrophoresis . If there is partial derivatisation you may get some labelled protein molecules to run into the gel. Others may not. There may be interaction between the labelled protein molecules causing precipitation.
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