Dbco sulfo cy5

Non-functionalized and azide-functionalized tri-ethylene glycol-substituted polyisocyanopeptide (PIC) polymers were synthesized as previously described (28 (link), 29 (link)). Briefly, non-functionalized isocyano-(D)-alanyl-(L)-alanine monomer and azide-terminated monomer mixed at a molar ratio of 1:30 were dissolved in toluene. After adding nickel percholate (Ni(ClO₄)₂₎ as a catalyst (catalyst-to-monomer molar ratio of 1:1,000), the reaction mixture was stirred at room temperature for 72 h. The reaction product was precipitated three times from dichloromethane in di-isopropyl ether. To produce GRDGS-functionalized PIC polymers (RGD-PIC), a solution of DBCO-NHS in DMSO was mixed with GRGDS peptide dissolved in borate buffer (pH 8.4) at 2 mg/mL and stirred for 3 h at room temperature. Mass spectrometry was performed to confirm the formation of DBCO-GRGDS conjugates. DBCO-GRGDS peptide was conjugated to azide-functionalized PIC polymers via strain-promoted azide-alkyne cycloaddition at a ratio of one DBCO-GRGDS per 100 monomers (28 (link)). DBCO-sulfo-Cy5 (Jena Bioscience) at a ratio of one DBCO-sulfo-Cy5 per 5,000 monomers was conjugated to the azide-functionalized PIC polymers in a similar manner to generate fluorescent PIC polymers.

The biosynthesis and purification of CK2α-pAzF was performed as described before [42 (link)]. For the recombinant expression of CK2β^1-193-pAzF, plasmids pCK2β^{1-193,Y176Stop} and pEVOL-pAzF were used to transform E. coli BL21(DE3). CK2β^1-193-pAzF was obtained and purified referring to the process of CK2α-pAzF [42 (link)] with the exception that before purification the cell lysate was stirred overnight at 4 °C in order to extract CK2β^1-193-pAzF [54 (link)].
Purified CK2α-pAzF (130 µg/mL) in buffer P50 (25 mM Tris/HCl (pH 8.5), 50 mM NaCl) was incubated with 50 µM DBCO-Sulfo-Cy5 (Jena Bioscience, Jena, Germany) for 1 h in the dark at room temperature (RT). By SPAAC reaction the specific labeled CK2α-DBCO-Sulfo-Cy5 was obtained. For the site-specific labeling of CK2β^1-193, purified CK2β^1-193-pAzF in buffer P100 (25 mM Tris/HCl (pH 8.5), 100 mM NaCl) was treated with fluorescein alkyne (0.25 mM), TCEP (Tris(2-carboxyethyl)phosphine, 1 mM), TBTA (Tris(benzyltriazolylmethyl)amine, 0.17 mM) and CuSO₄ (1 mM) for for 1 h in the dark at RT. By CuAAC reaction the specific labeled CK2β^1-193-Flu was obtained. For MST measurements an additional ultrafiltration step using vivaspin500 columns (Sartorius, Göttingen, Germany) was used to remove unbound fluorophore and additives of the click reaction.

The antibody was transferred into 0.1 M sodium bicarbonate at pH 8.3 and simultaneously concentrated by washing 3 times with 650 μl of buffer solution on a Spin-X UF 30K MWCO column (Corning). Different excesses of STP-N3 were added to 25 nmol of antibody, and the reaction mixtures were incubated at room temperature for 1 h. The excess reagent was removed using CentriPure MINI PBS Z-50 Spin Columns. The activity of the antibodies after azidation was examined using ELISA. The completeness of the reaction was controlled as follows. An aliquot of modified antibody was mixed with a tenfold molar excess of dibenzocyclooctyne derivative of sulfo-Cy5 (DBCO-Sulfo-Cy5, Jena Bioscience), followed by purification using gel filtration. The labeling level was calculated by analyzing the UV-vis spectra.