Total protein quantitation using the bicinchoninic acid (BCA) assay and gradient elution moving boundary electrophoresis (GEMBE)

Published

September 8, 2021

Author(s)

Jason Kralj, Matt S. Munson, David J. Ross

Abstract

We investigated using gradient elution moving boundary electrophoresis (GEMBE) and capacitively- coupled contactless conductivity detection (C4D) to assay total protein concentration using the BCA reaction. We chose this format because GEMBE is concentration dependent (unlike optical methods that rely on pathlength), tolerate particulates well that might otherwise scatter light, and can be multiplexed. The reaction was modified by reducing the pH slightly from 11.25 to 9.4, which allowed tartrate to be eliminated from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and capillary zone electrophoresis systems utilizing C4D. Under conditions similar to the "micro BCA" assay, we determined the limit of detection for three common proteins (insulin, bovine serum albumin, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact LOD and found that the conversion was proportional to the log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods.

Citation

Electrophoresis

Pub Type

Journals

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Keywords

Total protein assay, gradient elution moving boundary electrophoresis, bicinchoninic acid

Citation

Kralj, J. , Munson, M. and Ross, D. (2021), Total protein quantitation using the bicinchoninic acid (BCA) assay and gradient elution moving boundary electrophoresis (GEMBE), Electrophoresis, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=914716 (Accessed October 27, 2025)