Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates
| Autor: | Octavio T. Ramírez, Norma A. Valdez-Cruz, Mauricio A. Trujillo-Roldán, Saumel Pérez-Rodriguez |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine lcsh:Biotechnology Proteomics 01 natural sciences Applied Microbiology and Biotechnology Cell homogenates Precipitation protocols 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology lcsh:TP248.13-248.65 Acetone Protein precipitation Sample preparation Trichloroacetic acid lcsh:QH301-705.5 Chromatography Chinese hamster ovary cell 030104 developmental biology chemistry Protein recovery lcsh:Biology (General) Cell culture Chinese hamster ovary cells Protein solubilization Biotechnology Homogenization (biology) |
| Zdroj: | Electronic Journal of Biotechnology, Vol 48, Iss, Pp 86-94 (2020) |
| ISSN: | 0717-3458 |
| Popis: | Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins ( |
| Databáze: | OpenAIRE |
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