Uvod: Dvodimenzionalna gel-elektroforeza (2-DE) metoda je analize kompleksnih proteinskih smjesa izdvojenih iz stanica, tkiva, tjelesnih tekućina ili drugih bioloških uzoraka, koja objedinjuje dva različita elektroforetska postupka: izoelektrično fokusiranje i SDS-poliakrilamidnu gel-elektroforezu.
Metode: U ovom radu ispitan je utjecaj četi riju različiti h pufera na proteomske profile dviju staničnih linija karcinoma kolona, SW 620 i HCT 116 uzgojenih klasičnim metodama
uzgoja stanica in vitro uz pomoć metode 2-DE, u pH rasponu 3 – 10 NL, te rasponu molekulskih
težina od 14 do 100 kDa.
Rezultati : Dobiveni rezultati pokazuju kako se pojedini puferi
međusobno razlikuju po prirodi proteina koji su specifi čno otopljeni, što je djelomično ovisilo i o staničnoj liniji.
Diskusija: Topljivost proteina jedan je od najvećih problema pri korištenju 2-DE, što naročito dolazi do izražaja kod hidrofobnih membranskih proteina, proteina jezgre i proteina izrazito sklonih agregaciji. Jedan od načina poboljšanja topljivosti proteina je i opti mizacija pufera za izdvajanje proteina, pri čemu se u pufer dodaju zwitt er-ionski amfi fi lni surfaktanti poput CHAPS, SB 3-10 (kaprilil sulfobetain) i ASB-14, te uporabom ti ouree u kombinaciji s ureom. Gelovi dobiveni uz pomoć pufera koji sadrži CHAPS imali su najbolju rezoluciju i na njima je bilo moguće detekti rati najveći broj jedinstvenih proteinskih vrsta obiju ispitanih staničnih linija.
Zaključak: Postupkom opti mizacije pripreme uzorka za analizu mehanizama učinka novosinteti ziranog protutumorskog spoja na razini proteoma uz pomoć 2-DE pokazano je kako je pufer sastava 7M urea + 2M ti ourea + 4% (w/v) CHAPS opti malan pufer za izolaciju i razdvajanje smjese proteina dobivene iz tumorskih staničnih linija kolona HCT-116 i SW 620.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
