A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

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The Bicinchoninic Acid (BCA) Assay for Protein Quantitation

Springer Protocols Handbooks - The Protein Protocols Handbook ◽

10.1007/978-1-60327-259-9_3 ◽

1996 ◽

pp. 11-14 ◽

Cited By ~ 8

Author(s):

John M. Walker

Keyword(s):

Bicinchoninic Acid ◽

Protein Quantitation ◽

Bca Assay

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Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times

Analytical Biochemistry ◽

10.1016/j.ab.2020.113904 ◽

2020 ◽

Vol 608 ◽

pp. 113904 ◽

Cited By ~ 1

Author(s):

Javiera Cortés-Ríos ◽

Ana María Zárate ◽

Juan David Figueroa ◽

Joaquín Medina ◽

Eduardo Fuentes-Lemus ◽

...

Keyword(s):

Protein Quantification ◽

Bicinchoninic Acid ◽

Bca Assay

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

Nano LIFE ◽

10.1142/s1793984418500058 ◽

2018 ◽

Vol 08 (03) ◽

pp. 1850005 ◽

Cited By ~ 3

Author(s):

Daniel L. Smith ◽

Elizabeth N. Lemieux ◽

Sutapa Barua

Keyword(s):

Incubation Time ◽

Protein Concentration ◽

Coupling Reaction ◽

Careful Analysis ◽

Accurate Analysis ◽

Formation Reaction ◽

Bicinchoninic Acid ◽

Microparticle Formation ◽

Polymer Microparticles ◽

Bca Assay

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.

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