Improving SDS-PAGE method for monoclonal antibodies: The advantages of Tris-Acetate over Tris-Glycine SDS-PAGE system and comparison with CE-SDS method

Sonboli, Rozhan; Najafi, Zeinab; Zarezadeh, Nahid; Yazdani, Mohsen; Behrouz, Hossein

doi:10.1016/j.pep.2021.105845

Cited by 7 publications

(4 citation statements)

References 10 publications

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“…The higher voltage may cause an increase in heat and acceleration in the denaturation process. Better denaturation of proteins results in higher resolution, which can be the reason for a more broad range of molecular weight proteins when 110 V was applied than 80 V . According to these results, the H.…”

Section: Resultsmentioning

confidence: 89%

“…Better denaturation of proteins results in higher resolution, which can be the reason for a more broad range of molecular weight proteins when 110 V was applied than 80 V. 63 According to these results, the H. scoparia protein extracts were composed of nine protein bands with apparent molecular weights between approximately 135 and 17 kDa. In the study of Rouxel et al, 64 proteins of Palmaria palmata had six protein bands with apparent molecular weights between 59.6 and 15.2 kDa.…”

Section: 5mentioning

confidence: 92%

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Optimization of Protein Extraction from Halopteris scoparia Macroalgae by Ultrasonic-Assisted Enzymatic Extraction (UAEE): Bioactive, Chemical, and Technological Properties

Yücetepe,

Aydar,

Dogu-Baykut

et al. 2024

ACS Food Sci. Technol.

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Finding alternative food sources is now more crucial than ever because of the ever-growing global population, on the one hand, and the changing climate brought on by global warming, on the other. Macroalgae are currently a viable, novel, and alternative food source for the food industry. This research was centered on the optimization of extraction conditions of protein extracts from Halopteris scoparia by the response surface methodology based on the Box−Behnken design. According to the results, extraction yield (18.31−94.5%), total phenolic content (TPC) (11.70−19.07 mg GAE/g dw), and antioxidant activity (AOA CUPRAC : 11.97−17.85 mg TE/g dw, AOA ABTS : 45.45−78.02 mg TE/g dw), which were influenced by extraction parameters, were investigated. The optimum extraction conditions were ultrasound application time of 7.46 min, enzyme/substrate of 0.8, and extraction time of 2 h. The water (0.29 ± 0.07 g/g) and oil absorption capacity (1.37 ± 0.06 g/g), foaming capacity (8.50 ± 3.70%) and stability (8.50 ± 3.70%), and emulsion activity (37.5 ± 0.00%) and stability (62.22 ± 7.70%) of the protein extracts obtained under optimum conditions were investigated along with Fourier transform infrared spectroscopy, differential scanning calorimetry, and SDS-PAGE. Protein extracts obtained from H. scoparia showed high TPC and AOA, while the emulsion and foaming properties were found to be lower than those of the algal or plant proteins. The hydrolysis degree of protein hydrolysate from H. scopariaobtained enzymatic hydrolysis was found to be 61.43%. Sixteen amino acids were identified from the H. scoparia protein hydrolysate. The total amino acid content in the protein hydrolysate was 10.44 mg/g protein. Tyrosine, one of the nonessential amino acids, and leucine and lysine, two of the essential amino acids, were determined as the main amino acids.

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Section: Resultsmentioning

confidence: 89%

Section: 5mentioning

confidence: 92%

Optimization of Protein Extraction from Halopteris scoparia Macroalgae by Ultrasonic-Assisted Enzymatic Extraction (UAEE): Bioactive, Chemical, and Technological Properties

Yücetepe,

Aydar,

Dogu-Baykut

et al. 2024

ACS Food Sci. Technol.

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“…Moreover, proline content of the analyte also has critical impact on electrophoretic behavior which may result in a higher apparent MW [8]. Thus, the apparent molecular mass of proteins identified by SDS-PAGE may not be a true reflection of the mass of polypeptide chain [7,19]. As reported by Dong and Kang, advanced and automated versions of this approach, such as CE-SDS technology, could be employed as a promising alternative to obtain higher resolution and better precision about MW of complex proteins [20].…”

Section: Sds-pagementioning

confidence: 99%

“…Determination of MW of proteins after treating them with a strong anionic detergent, SDS, and separation based on migration distance in PAGE, is a well‐established method [7]. Since its introduction in 1969, this is still the widely used method because of its simplicity, rapidity, and inexpensive nature.…”

Section: Introductionmentioning

confidence: 99%

A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation

et al. 2022

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Biotherapeutics, such as mAbs and fusion proteins, are a major and rapidly growing class of pharmaceuticals. Majority of the biopharmaceuticals are glycoproteins, wherein about 1 to 30% of their molecular weight (MW) are contributed by the glycans. Determination of MW of heavily glycosylated proteins, such as Fc-fusion proteins, is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation influences the expected size of the glycoprotein, which leads to disproportionate MW estimation, in size-dependent methods. Hence, in this study, we have demonstrated the advantages and limitations of four widely used MW estimation techniques for three proteins having varying levels of glycosylation. It was proven that glycosylation had least impact on MW determination by SEC-MALS and SV-AUC. However, MW estimation by LC-MS and SDS-PAGE was extensively hampered by the degree of glycosylation. It is, thus, essential to consider the structural characteristics of proteins while selecting a technique for determining their MW.

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Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology‐based products

et al. 2021

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Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology-based productsBiopharmaceuticals (recombinant technology-based products, vaccines, whole blood and blood components, gene therapy, cells, tissues, etc.,) are described as biological medical products produced from various living sources such as human, microbial, animal, and so on by manufacturing, extraction, or semi-synthesis. They are complex molecules having high molecular weights. For their safety and efficacy, their structural, clinical, physicochemical, and chemical features must be carefully controlled, and they must be well characterized by analytical techniques before the approval of the final product. Capillary electrophoresis (CE) having versatile modes can provide valuable safety and efficacy information, such as amino acid sequence, size variants (low and high molecular weight variants), charged variants (acidic and basic impurities), aggregates, N-linked glycosylation, and O-linked glycosylation. There are numerous applications of CE in the literature. In this review, the most significant and recent studies on the analysis of recombinant DNA technology-based products using different CE modes in the last ten years have been overviewed. It was seen that the researches mostly focus on the analysis of mAbs and IgG. In addition, in recent years, researchers have started to prefer CE combined mass spectrometry (MS) techniques to provide a more detailed characterization for protein and peptide fragments.

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Improving SDS-PAGE method for monoclonal antibodies: The advantages of Tris-Acetate over Tris-Glycine SDS-PAGE system and comparison with CE-SDS method

Cited by 7 publications

References 10 publications

Optimization of Protein Extraction from Halopteris scoparia Macroalgae by Ultrasonic-Assisted Enzymatic Extraction (UAEE): Bioactive, Chemical, and Technological Properties

Optimization of Protein Extraction from Halopteris scoparia Macroalgae by Ultrasonic-Assisted Enzymatic Extraction (UAEE): Bioactive, Chemical, and Technological Properties

A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation

Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology‐based products

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