A visual detection of protein content based on titration of moving reaction boundary electrophoresis

Wang, Hou-Yu; Guo, Cheng-ye; Guo, Chaowan; Fan, Liu-Yin; Zhang, Lei; Cao, Cheng-Xi

doi:10.1016/j.aca.2013.03.008

Cited by 18 publications

(33 citation statements)

References 32 publications

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“…Then in 2013, Wang et al . and Guo et al . advanced the MRBT technique for determination of total protein content in milk.…”

Section: Introductionmentioning

confidence: 90%

“…Based on these concepts, Yang et al [22] and Wang et al [23] developed the MRBT method and designed the relevant device for acid-base titration analysis. Then in 2013, Wang et al [24] and Guo et al [25] advanced the MRBT technique for determination of total protein content in milk. In this MRBT system, the MRB is formed by hydroxide ions in the catholyte and acidic residues of milk proteins immobilized in highly cross-linked polyacrylamide gel, and an acid-base indicator is used to denote the boundary movement.…”

Section: Introductionmentioning

confidence: 99%

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Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration

Luo

Zheng

Chen

et al. 2018

Biochem Molecular Bio Educ

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Laboratory exercises focused on protein quantification are frequently conducted in traditional undergraduate biochemistry laboratory curriculum. The laboratory course described here is designed to provide students with experience in measurement of protein content in milk powder by moving reaction boundary titration (MRBT), a new rapid technique for total protein content determination in milk. In addition, this approach is weakly influenced by nonprotein nitrogen reagents such as melamine and urea. The course was done as three weekly laboratory exercises. First, students established a standard curve for milk protein concentration by MRBT method. Then, students investigated the influence of nonprotein nitrogen reagents on MRBT method. Finally, students made a comparison among three different protein quantification methods (MRBT, Biuret, and Kjeldahl method). From the experiments, students grasped the concept and advantages of MRBT and deepened the understanding of protein quantification. This course offer students the opportunity to be exposed to an advanced technique, which may have practical significance to their future study and work in the life science field. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):644–651, 2018.

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“…Then in 2013, Wang et al . and Guo et al . advanced the MRBT technique for determination of total protein content in milk.…”

Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration

Luo

Zheng

Chen

et al. 2018

Biochem Molecular Bio Educ

View full text Add to dashboard Cite

show abstract

“…To address the issue of NPN adulteration in milk products, the concept of moving reaction boundary titration (MRBT) was proposed, based on the moving reaction boundary (MRB) electrophoresis . In the MRBT analysis, samples or milk proteins were immobilized in highly cross‐linked polyacrylamide gel and hydrogen or hydroxide ions could migrate into the gel and neutralized the alkaline or acidic groups of proteins under electric field . After that, protein content of milk could be measured by the movement of neutralization boundary under a certain running time.…”

Section: Introductionmentioning

confidence: 99%

“…After that, protein content of milk could be measured by the movement of neutralization boundary under a certain running time. MRBT method could accurately monitor protein content and averted the bias influence from NPN by avoiding NPN titration in milk . Recently, the concept of retardation signal of reaction boundary electrophoresis was proposed, manifesting a linearity between the boundary velocity and the protein content, greatly simplified the analysis of protein content in milk samples ; the lever principle of double protein titration was revealed for analyzing poor protein adulteration ratio in good milk or food according to the retardation signal concept .…”

Section: Introductionmentioning

confidence: 99%

“…Since, chip‐based titration techniques are still suffered from instability due to the poor control of electro‐osmotic flow , it is still challenging for establishing a mature application of electrophoretic titration chip. A stable run of MRBT by using the previous titration device was mainly implemented with double four‐way tube electrode chambers . However, currently used device is suffered from the electrolyte leakage (Supporting Information Fig.…”

Section: Introductionmentioning

confidence: 99%

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A stable and convenient protein electrophoresis titration device with bubble removing system

Zhang

Fan

et al. 2017

Electrophoresis

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Moving reaction boundary titration (MRBT) has a potential application to immunoassay and protein content analysis with high selectivity. However, air bubbles often impair the accuracy of MRBT, and the leakage of electrolyte greatly decreases the safety and convenience of electrophoretic titration. Addressing these two issues a reliable MRBT device with modified electrolyte chamber of protein titration was designed. Multiphysics computer simulation was conducted for optimization according to two-phase flow. The single chamber was made of two perpendicular cylinders with different diameters. After placing electrophoretic tube, the resident air in the junction next to the gel could be eliminated by a simple fast electrolyte flow. Removing the electrophoretic tube automatically prevented electrolyte leakage at the junction due to the gravity-induced negative pressure within the chamber. Moreover, the numerical simulation and experiments showed that the improved MRBT device has following advantages: (i) easy and rapid setup of electrophoretic tube within 20 s; (ii) simple and quick bubble dissipates from the chamber of titration within 2 s; (iii) no electrolyte leakage from the two chambers: and (iv) accurate protein titration and safe instrumental operation. The developed technique and apparatus greatly improves the performance of the previous MRBT device, and providing a new route toward practical application.

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Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis

2014

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We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to 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.

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A visual detection of protein content based on titration of moving reaction boundary electrophoresis

Cited by 18 publications

References 32 publications

Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration

Undergraduate laboratory experiment on determination of total protein content in milk powder by moving reaction boundary titration

A stable and convenient protein electrophoresis titration device with bubble removing system

Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis

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