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J Ind Microbiol Biotechnol (2016) 43:12711280 DOI 10.1007/s10295-016-1796-9
FERMENTATION, CELL CULTURE AND BIOENGINEERING
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Web End = Bioprocess monitoring: minimizing sample matrix effects for total protein quantication with bicinchoninic acid assay
Wieland N. Reichelt1 Daniel Waldschitz1 Christoph Herwig1,2 Lukas Neutsch2
Abstract Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the eld of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufcient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A signicant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modication of the BCA assay, which is less inuenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the
Received: 1 March 2016 / Accepted: 31 May 2016 / Published online: 17 June 2016 The Author(s) 2016. This article is published with open access at Springerlink.com
conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantication could be improved vefold, taking the BCA protein quantication to a level of accuracy comparable to other, more expensive methods.This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics.
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Electronic supplementary material The online version of this article (doi:http://dx.doi.org/10.1007/s10295-016-1796-9
Web End =10.1007/s10295-016-1796-9 ) contains supplementary material, which is available to authorized users.
* Christoph Herwig [email protected]
1 Christian Doppler Laboratory for Mechanisticand Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna Universityof Technology, Getreidemarkt 9/166, 1060 Vienna, Austria
2 Research Division Biochemical Engineering, Instituteof Chemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1A/166-4, 1060 Vienna,...