IQB201 Aula 05 Metodos para Purificacao de Proteinas 03 2011

IQB201 – Bioquímica Básica I

Purificação de Proteínas
Joab Trajano Silva
Instituto de Química/UFRJ

As diferenças de características das proteínas (como tamanho, ponto isoelétrico e atividade biológica) podem ser usadas para promover a purificação de uma proteína de interesse a partir de uma mistura complexa.

Salting Out

Representação da superfície de uma proteína. As áreas vermelhas representam resíduos com cargas negativas, as áreas em azul representam resíduos com carga positiva. Quando as proteínas se enovelam, os resíduos hidrofóbicos tendem a ficar no seu interior. Entretanto, sempre ficam "patches“ hidrofóbicos expostos na superfície. Cromatografia Líquida

Cromatografia de Exclusão em Gel

N-acetylglucosamine-6-sulphatase

(A) Acid extracts of granules obtained from human granulocytes were loaded on a Sephadex G-75 column (2.5 cm×90 cm) and eluted with 0.2 M sodium acetate, pH 4.5, as described in the Experimental section, giving peaks A–E. As indicated, the fractions in the peaks (A–E) were pooled (pools 1–5). The majority of the 75 kDa protein was contained in pool 2(a) of peak B
(fractions 68–76), as indicated by the horizontal bar, as judged by SDS/PAGE after further separation of proteins in each pool on a Mono-S column (results not shown). Biochemical Journal (2005) Volume 387, 841-847

Cromatografia de Troca Iônica

Cromatografia de Troca Iônica

Cromatografia de Troca Iônica

Cromatografia de Troca Iônica

Figure 1 Separation of granule proteins by gel filtration and ion-exchange chromatography. (B) Ion-exchange chromatography was performed as described in the Experimental section. Fractions 68–76 from the gel filtration column were applied to the Mono-S column and eluted by a linear gradient from 0 to 1.0 M NaCl in
0.1 M sodium acetate, pH 4.0. The 75 kDa protein was eluted in fractions 11–17 of peak 2, as indicated by the horizontal bar. Biochemical Journal (2005) Volume 387, 841-847

Cromatografia de Afinidade