Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

Sissolak, Bernhard; Zabik, Christian; Saric, Natasa; Sommeregger, Wolfgang; Vorauer-Uhl, Karola; Striedner, Gerald

doi:10.1002/biot.201800714

Cited by 18 publications

(11 citation statements)

References 36 publications

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“…The reducing sugar content of the Se−TPSs was determined by the dinitrosalicylic acid method, using glucose as the standard [ 24 ]. The protein content was measured by the Bradford method, using bovine serum albumin as the standard [ 25 ]. The Folin–Ciocalteau colorimetric method was used to determine the total polyphenols in the Se−TPS samples [ 26 ].…”

Section: Methodsmentioning

confidence: 99%

Polysaccharides in Selenium-Enriched Tea: Extraction Performance under Innovative Technologies and Antioxidant Activities

Gao

Zhang

et al. 2022

Foods

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Pulsed electric fields (PEF) and ultrasonic-assisted extraction (UE) were applied to improve the extraction performance of selenium-enriched tea polysaccharides (Se−TPSs) in mild conditions. Two combined extraction processes were investigated: (1) PEF strength at 10 kV/cm followed by conventional extraction (CE) at 50 °C for 60 min and (2) PEF+UE (PEF strength at 10 kV/cm followed by UE at 400 W for 60 min). The optimal extraction yields, and energy consumption rates were obtained at 36.86% and 41.53% and 78.78 kJ/mg and 133.91 kJ/mg, respectively. The Se−TPSs were analyzed and characterized by GPC, UV, and FT-IR, which evidenced the structural stability of the Se−TPSs during the extraction processes. It was found that PEF and UE could reduce the particle size diameter of the Se−TPS extract, as well as the proportion of uronic acid. Moreover, PEF could increase the selenium content in the Se−TPS extract by 160.14% due to a lower extraction temperature compared to conventional extraction. The antioxidant activities of the Se−TPSs in vitro were investigated using OH, O2−, and ABTS+ scavenging experiments, as well as a total antioxidant ability evaluation. It was found that the antioxidant activity of the Se−TPSs obtained using PEF2+CE2 was relatively high due to the potential synergistic effect between the selenium and polysaccharides. Based on these results, we speculate that PEF2+CE2 was the best extraction process for the Se−TPSs. Furthermore, this research indicates the application of selenium-enriched tea for functional food production.

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Section: Methodsmentioning

confidence: 99%

Polysaccharides in Selenium-Enriched Tea: Extraction Performance under Innovative Technologies and Antioxidant Activities

Gao

Zhang

et al. 2022

Foods

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“…After centrifugation, the urine was placed in a 1.5 mL EP tube and stored at −80°C. Urine protein content was determined by Bradford method [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

Gypenosides regulate autophagy through Sirt1 pathway and the anti-inflammatory mechanism of mitochondrial autophagy in systemic lupus erythematosus

Liu

Wang

et al. 2022

Bioengineered

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To study the mechanism of gynostemma pentaphyllum saponins (GpS) regulating mitochondrial autophagy and anti-inflammatory through Sirtuin 1 (Sirt1) pathway in systemic lupus erythematosus (SLE). JURKAT cells were cultured in vitro, RT-PCR and western blotting (WB) were utilized to identify the expression of related-proteins in Sirt1 pathway and global autophagy and mitochondrial autophagy markers in JURKAT before and after GpS treatment induced by ultraviolet B (UVB), and the related-mechanism of GpS regulation of autophagy was analyzed. The SLE model was established to analyze the alleviating effects of GpS on various symptoms of lupus mice. Sirt1/AMPK/mTOR pathway was activated in UVB induced JURKAT cells. After the addition of GpS, WB revealed that the phosphorylation of AMPK decreased, the phosphorylation of mTOR increased, the expression of Sirt1 protein decreased, and the activation of the pathway was inhibited. Moreover, autophagy of JURKAT cells wasinhibited. In order to further verify the role of Sirt1 pathway, we activated Sirt1 expression in cells by constructing lentiviral vectors, and the therapeutic effect of GpS was significantly reduced. These results indicate GpS can exert autophagy regulation by inhibiting the activity of Sirt1 pathway. To treat SLE. GpS can significantly reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice. GpS can regulate autophagy and mitochondrial autophagy through Sirt1 pathway, which may be a potential mechanism for GpS to reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice.

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“…Product titer in culture supernatants was determined by bio‐layer interferometry (BLI) with the Octet QK system (Pall Forte Bio), as previously described by Sissolak et al 16 Briefly, samples were diluted in phosphate buffered saline with 0.1 vol% Tween 20 (PBS‐T) and transferred to each well of a black, nonsterile 96‐well plate (Thermo Fischer Scientific). Protein A biosensor tips were equilibrated in PBS‐T, and plates were shaken at 1000 rpm for 5 s before each measurement.…”

Section: Methodsmentioning

confidence: 99%

“…The mock control experiment was performed with the nonproducing host cell line and was carried out in a shake flask, as described by Sissolak et al 16 3 | RESULTS AND DISCUSSION…”

Section: Bioprocess Designmentioning

confidence: 99%

Quantification of glycated IgG in CHO supernatants: A practical approach

Lhota

Sissolak²,

Striedner

et al. 2021

Biotechnology Progress

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Post-translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process-design considerations, especially concerning glucose-feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean-up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira®, was successfully applied, and continuous monitoring of mAbs across three repetitive fed-batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch-consistency monitoring.

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Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

Cited by 18 publications

References 36 publications

Polysaccharides in Selenium-Enriched Tea: Extraction Performance under Innovative Technologies and Antioxidant Activities

Polysaccharides in Selenium-Enriched Tea: Extraction Performance under Innovative Technologies and Antioxidant Activities

Gypenosides regulate autophagy through Sirt1 pathway and the anti-inflammatory mechanism of mitochondrial autophagy in systemic lupus erythematosus

Quantification of glycated IgG in CHO supernatants: A practical approach

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