Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates
Electron. j. biotechnol
;
48: 86-94, nov. 2020. tab, graf, ilus
Artículo
en Inglés
| LILACS
| ID: biblio-1254836
ABSTRACT
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 (< 15â¯kDa).CONCLUSIONS:
These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.
Texto completo:
Disponible
Índice:
LILACS (Américas)
Asunto principal:
Proteínas Recombinantes
/
Células CHO
/
Proteómica
Tipo de estudio:
Guía de Práctica Clínica
Límite:
Animales
Idioma:
Inglés
Revista:
Electron. j. biotechnol
Asunto de la revista:
Biotecnologia
Año:
2020
Tipo del documento:
Artículo
País de afiliación:
México
Institución/País de afiliación:
Universidad Nacional Autónoma de México/MX
Similares
MEDLINE
...
LILACS
LIS