The PSarRFP reaction mixture is represented with two clear signals attributed to excessed PSaroxid and conjugated RFP (Figure , dark blue). This unexpected observation could be related either to inferior conjugation degree or decreased PSarRFP hydrodynamic radius after functionalization. A noticeable decrease of the second peak and increased molecular weight shoulder after centrifugation (most molecules lower than 10.0 kg/mol were removed) are observed (Figure , blue). Side reactions could also explain the presence of the high molecular shoulder in PSarRFP traces during conjugation. PAGE analysis shows that electrophoretic patterns of RFP alone exhibit a characteristic band at ca. 25 kg/mol while the conjugated PSarRFP before centrifugation shows three prominent bands that could be attributed to free RFP and the desired PSarRFP that appeared at lower Mw, which suggesting that presence of PBS increases the migration rate of PSarRFP (Figure ). PAGE of the purified PSarRFP shows only one band at the same region that native RFP and the presence of minor smear at higher Mw was not detected (Figure ). We expect that reaction conditions critically influence the degree of conjugation and that further improvements are needed. BSA was selected as another model protein for conjugation due to its ready availability, robust nature, and the presence of an accessible free thiol at Cys-34 and 60 free amine groups available for conjugation . The conjugation method uses a large excess of PSaroxid (Mw = 3.5 kg/mol). The experiment was carried out for 5 h and 72 h in PBS at pH = 8.0. Subsequently, the reaction mixture was dialyzed against water for 4 h to remove unreacted polymer before lyophilization. GPC analysis shows a minor shift after 5 h and a significant tailing at higher molar mass after 72 h (Figure ). The negligible molar mass difference between PSarBSA and native BSA suggests that the conjugation via the amine group is avoided under these pH conditions, and only cysteine group is available for interaction. PSarBSA incubated for 72 h shows higher molecular weight tailing compared with native BSA and, also, presence of low molecular weight tailing for signal attributed to PSaroxid (Figure ). The presence of tailing points that increased incubation time leads to increased side reactions with PSaroxid like chain coupling or degradation and increases the degree of conjugation (Figure ). The SDS-PAGE analysis of PSarBSA performed after coupling in PBS at pH = 8.5 (PSarBSA1) and 11 (PSarBSA2) demonstrate a successful mere successful result. A small but distinct increase in molar mass was found between BSA and its corresponding conjugates (PSarBSA1 and PSarBSA2), respectively. The average molar mass was estimated to be ca. 75-80 kg/mol (supporting information Figure SI). Our results suggest that the degree of conjugation depends on the pH of the reaction media (Figure ), which is well known for the reaction of activated carboxylic acids. The difference in band intensity points that significant excess of unreacted PSar is present even after 24h of dialysis (membrane pore size is 10 kDA).