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 NN-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 μg/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 20min, compared with the recommended 30min. This re-assessment of BCA assay is important to avoid misestimating biases in bioconjugation processes.
D. L. Smith et al., "Correction in Bicinchoninic Acid (BCA) Absorbance Assay to Analyze Protein Concentration," Nano LIFE, World Scientific Publishing, Jun 2018.
The definitive version is available at https://doi.org/10.1142/S1793984418500058
Chemical and Biochemical Engineering
Keywords and Phrases
BCA assay; Compat-Able Protein Assay; gelatin; NHS interference
International Standard Serial Number (ISSN)
Article - Journal
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01 Jun 2018