Our lab has successfully designed a chimeric IL-15 protein by fusing IL-15 with the IgG2/IgG2a constant domain. We are now planning to study its biodistribution in mice. For this, we will tag the chimeric IL-15 with a non-toxic fluorescein dye, inject the conjugated protein into mice, and observe its distribution across various organs (e.g., liver, lungs, intestinal tissues etc.) at different time points (1 hr, 2 hr, 6 hr, 24 hr, 48 hr, etc.) using a live imaging system.
The molecular weight of our protein molecule is approximately 100 kDa as a dimer and 50 kDa as a monomer, while the dye used for tagging (VivoTag680XL) has a molecular weight of 1.8 kDa. We follow the tagging protocol provided with the kit, and unconjugated fluorophores are removed using purification columns with a molecular weight cutoff of 7 kDa.
I seek your guidance on confirming the successful conjugation of the dye to the protein. Since there is a possibility of free dye remaining even after purification, is there a specific technique you would recommend for validating the coupling?
You could run the labeled protein on an analytical SEC column (e.g. Superdex 75, not very cheap). If the fluorescense co-migrates with the protein it is most likely covalently bound. The purification column uses the same principle but with a resin that has not a high resolution, so any molecule >7 kDa will pass through while the label migrates slower through the resin.
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