Medicina Fluminensis, Vol. 45 No. 3, 2009.
Izvorni znanstveni članak
Proteomics in drug development – optimizati on of sample preparation for the two-dimensional gel-electrophoresis
Mirela Sedić
; Insti tut “Ruđer Bošković”, Zavod za molekularnu medicinu, Laboratorij za sistemsku biomedicinu,, Zagreb, Hrvatska
Sandra Kraljević Pavelić
; Insti tut “Ruđer Bošković”, Zavod za molekularnu medicinu, Laboratorij za sistemsku biomedicinu,, Zagreb, Hrvatska
Sažetak
Introducti on: The two-dimensional gel-electrophoresis (2-DE) is a method of choice for complex protein mixture separati on from cells, ti ssue, body fl uids or other biological samples which comprises two diverse electrophoreti c principles: the isoelectric focusing
and the SDS-polyacrilamide gel electrophoresis.
Methods: In this paper we tested the effects of four diff erent 2-DE buff ers on the protein profi les obtained by 2-DE in the pH
range 3-10 NL, and the molecular weight range 14-100 kDa. The proteins were isolated from two colon cancer cell lines SW 620 i HCT 116 grown in vitro by using standard cell propagation procedures. Results: The obtained results revealed diff erent 2-DE buff er properti es for each buff er tested as the protein profi les showed a diff erent patt ern both as the consequence of the buff er type and the type of cell line.
Discussion: One of the major problems in 2-DE is protein solubility which is parti cularly emphasized when dealing with hydrophobic
membrane proteins, the nuclear proteins as well as with proteins prone to form aggregates. One of the possible soluti ons to this problem is the opti mizati on of the 2-DE buff er by additi
on of zwitt er-ionic amphyphilic surfactants like CHAPS, SB 3-10 (sulphobetain) and ASB-14, as well as by additi on of thiourea and urea. The gels obtained with the buff er containing CHAPS showed the best resoluti on properti es and had the best unique protein species score for both tested cell lines in comparison to all other gels.
Conclusion: Through the 2-DE buffer optimization in the sample preparati on step for proteomic analysis of newly synthesized
anti tumor drug mechanisms of acti on, it has been shown that the buff er containing 7M urea + 2M thiourea + 4% (w/v) CHAPS was the most eff ecti ve for isolati on and separati on of proteins
from HCT-116 and SW 620 tumour cell lines.
Ključne riječi
two-dimensional gel-electrophoresis; optimization of sample preparation; proteomics
Hrčak ID:
43670
URI
Datum izdavanja:
1.9.2009.
Posjeta: 2.987 *