KOSEN 한인과학기술자네트워크

>특정 단백질의 dimer를 형성하는 부위를 찾고자하여>이 특정 단백질의 일부분에대한 펩타이드를 합성하였습니다.>이 펩타이드를 특정 단백질과 in vitro 반응시킨후 결합하는지를 확인하고자 웨스턴 블럿을 하였습니다(reducing 과 non reducing condition). 펩타이드를 첨가하지 않은 것에서는 dimer가 형성되지 않은 반면, 펩타이드가 첨가된 것에서는 reducing condition에서도 특정단백질의 dimer가 형성이 되었습니다. >>특정 단백질의 dimer 형성이 어떻게 일어날 수 있는지?>그리고 이 dimer를 깰수 있는 agents가 어떤것이 있는지 궁금합니다.>>여러분의 고견을 부탁드립니다. DTT is an unusually strong reducing agent, owing to its high conformational propensity to form a six-membered ring with an internal disulfide bond. It has a redox potential of -0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The intermediate mixed-disulfide state is unstable (i.e., poorly populated) because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond. The reducing power of DTT is limited to pH values above ~7, since only the negatively charged thiolate form -S? is reactive (the protonated thiol form -SH is not); the pKa of thiol groups is typically ~8.3. TCEP (tris(2-carboxyethyl)phosphine) is a reducing agent frequently used in biochemistry and molecular biology applications[1]. It is often prepared and used as a hydrochloride salt. TCEP is also available as a stabilized solution at neutral pH and immobilized onto an agarose support to facilitate removal of the reducing agent. TCEP is often used as a reducing agent to break disulfide bonds within and between proteins as a preparatory step for gel electrophoresis. Compared to the other two most common agents used for this purpose (dithiothreitol and β-mercaptoethanol), TCEP has the advantage of being odourless, more powerful reductant, irreversible reductant, more hydrophilic and is more resistant to oxidation in air. TCEP also does not reduce metals used in immobilized metal affinity chromatography 2-Mercaptoethanol (also β-mercaptoethanol, or "2BME") is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol, HOCH2CH2OH, and 1,2-ethanedithiol, HSCH2CH2SH. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by scavenging hydroxyl radicals (amongst others) . It is widely used because the hydroxyl group confers solubility in water and lowers the volatility. Due to its diminished vapor pressure, its odour, while unpleasant, is less objectionable than related thiols.