Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Kurfürst, Manfred

doi:10.1016/0003-2697(92)90460-o

Cited by 202 publications

(144 citation statements)

References 12 publications

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“…Although PEG could be quantified in PEG-containing control samples, the PEG concentration within the final HEK293T-CD63-eGFP EV samples (clone B2 and C5 EVs were tested) was below the optimal detection range of this method (data not shown). Thus, a more sensitive barium-iodide staining technique [43] was selected to analyse HEK293T-CD63-eGFP EV samples.…”

Section: Resultsmentioning

confidence: 99%

“…After electrophoresis for 40 min at 200 V in MOPS-buffer (Thermo Fisher) gels were washed twice with distilled water. To visualise the PEG, gels were incubated for 10 min in freshly prepared 5% barium chloride (ROTH, Karlsruhe, Germany) and developed with 0.1 M iodide solution (ROTH, Karlsruhe, Germany) [43,44]. After the positive detection of the PEG signals, the solution was exchanged for distilled water.…”

Section: Methodsmentioning

confidence: 99%

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Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Ludwig

Miroschedji

Doeppner

et al. 2018

J of Extracellular Vesicle

190

206

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Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Ludwig

Miroschedji

Doeppner

et al. 2018

J of Extracellular Vesicle

190

206

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“…12 Los geles fueron teñidos específicamente para proteínas con Coomassie brilliant blue (CBB) y para PEG con yodo. 13 La imágenes digitales de los geles fueron obenidas con un escaner ScanJet 4c/T (Hewlett Packard, Palo Alto, CA, USA) y analizadas con el software Molecular Analyst (Bio-Rad Laboratory, Hercules, CA, USA).…”

Section: Electroforesis En Geles De Poliacrilamida Y En Presencia De unclassified

Un método reproducible para obtener peg biramificado monofuncional de alta pureza

Ramón¹,

Péniche²,

Castro³

et al. 2009

Quím. Nova

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Recebido em 28/5/08; aceito em 4/2/09; publicado na web em 3/7/09 A REPRODUCIBLE METHOD FOR OBTAINING A 2-ARMS MONOFUNCTIONAL PEG WITH HIGH PURITY. PEGylation has become a widely applied technique to enhancing in vitro and in vivo stability of therapeutic proteins and to increasing materials biocompatibility. PEG branched structures have proven useful for protein and peptide modification. Furthermore, they may be better than linear structures for many purposes. This paper describes an improved procedure for obtaining 2-arms PEG based on L-lysine. The efficiency of the synthesis was not related to moisture of the raw materials. This procedure does not use hazardous reagents as previous protocols do. It implemented a purification process for obtaining the desired structure with high purity (> 99%). Finally, the procedure described here allows the obtaining of others PEGylation reagents.Keywords: PEGylation; branched PEG; interferon. INTRODUCCIÓNEl polietilenglicol (PEG) es un homopolímero sintético obtenido a partir del óxido de etileno. La habilidad única del PEG de ser soluble tanto en agua como en solventes orgánicos permite que pueda ser funcionalizado fácilmente y que la conjugación química a biomoléculas pueda hacerse en condiciones suaves que no afecten a éstas. 1 Estudios del PEG en solución acuosa han demostrado que este polímero une 2-3 moléculas de agua por unidad de óxido de etileno. Debido a su alta flexibilidad y a la unión de las moléculas de agua el PEG actúa como si fuera 5-10 veces más grande que una proteína soluble de masa molecular comparable. Se ha sugerido que estas propiedades son las responsables de que el PEG: precipite las proteínas; excluya a las proteínas y células de las superficies; reduzca su inmunogenicidad y antigenicidad y prevenga la degradación de las proteínas por enzimas y células de mamíferos. 2 Debido a estas características, la conjugación a polietilenglicol, conocida como PEGilación, se usa ampliamente para: aumentar la estabilidad in vitro e in vivo de proteínas terapéuticas, disminuir la adsorción de proteínas y células en la superficie de biomateriales, aumentar el tiempo de vida en el organismo de liposomas, entre otras muchas aplicaciones. 2 Los primeros trabajos de PEGilación se realizaron en la década de los ´70. La posibilidad teórica de usar la conjugación a PEG de las proteínas, para disminuir la inmunogenicidad de éstas, se le ocurrió a Frank F. Davis en 1967 mientras preparaba la solicitud de financiamiento para un proyecto sobre aplicación terapéutica de proteínas no humanas. 3 No obstante, no es hasta inicios de los ´90 cuando se aprueba el uso en humanos de las primeras proteínas PEGiladas (Adenosin Deamidasa y Catalasa de Hígado Bovino). 4 Los estudios realizados durante los ´80 y los ´90 demostraron que la mayor utilidad de la PEGilación es en el aumento del tiempo de vida media en sangre de las proteínas conjugadas y no en la disminución de la inmunogenicidad de éstas.Existen diversos procedimientos para obtener reactivos para PEGilar con variadas estruc...

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“…Our results indicate that large proportional PEGylated protein could not be obtained by these acetonitrile is efficient to enhance the resolution of methods [8][9][10]. Sodium dodecyl sulfate-polyPEGylated proteins, while the total analysis time is acrylamide gel electrophoresis (SDS-PAGE) [11], reduced compared to the reported methods. high-performance liquid chromatography (HPLC)…”

Section: Introductionmentioning

confidence: 97%

Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis

Zhong

Lin

et al. 2001

Journal of Chromatography A

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Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Cited by 202 publications

References 12 publications

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Un método reproducible para obtener peg biramificado monofuncional de alta pureza

Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis

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