Correction in Bicinchoninic Acid (BCA) Absorbance Assay to Analyze Protein Concentration

Conducting the bicinchoninic acid (BCA) assay directly after a coupling reaction using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and [Formula: see text]-hydroxysuccinimide (NHS) chemistry produces significant errors. Here we present a correction for the quantification of gelatin in the supernatant (SN) following gelatin conjugation to polymer microparticles using EDC and NHS chemistry. Following the conjugation reaction, SNs from the gelatin-microparticle formation reaction are treated with BCA assay reagents and quantified for the percentage of unbound gelatin in the solution. NHS was found to interfere with the BCA assay reagents and is dependent on incubation time. It is found that the large concentration (500[Formula: see text][Formula: see text]g/mL) of NHS in the conjugation reaction interferes with the sensitivity of gelatin present in SNs. The interference from NHS requires a careful analysis to distinguish the BCA background absorbance from the sample absorbance. Using an NHS control solution can correct NHS interference and thus decrease the expensive iterations in gelatin quantification and enable accurate analysis of gelatin content. The accuracy of gelatin quantification is further improved by reducing the BCA assay incubation time to approximately 20[Formula: see text]min, compared with the recommended 30[Formula: see text]min. This re-assessment of BCA assay is important to avoid misestimating biases in bioconjugation processes.