purificao das proteinas
TRANSCRIPT
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Purificação de proteínas
ETAPAS PRÉVIAS – DECIDIR:
Fonte de proteína
Método para dosar a atividade biológica
Método para dosar a quantidade total de proteínas
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Extrato bruto
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Extrato bruto
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Centrifugação
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Centrifugação
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Centrifugação
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Purificação de proteínas
solubilidadetamanhocargaafinidade de ligação
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Purificação de proteínas
CADA ETAPA DA PURIFICAÇÃO:
TESTAR A ATIVIDADE BIOLÓGICA
DETERMINAR CONCENTRAÇÃO DE PROTEÍNAS
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SOLUBILIDADE
Solubilidade
Força iônica (M)
“Salting in”
“Salting out”
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Exemplos de fracionamento “salting out”
Fibrinogênio = 0,8 M de (NH4)2SO4- Albumina = 2,4 M de (NH4)2SO4
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“Salting out”
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Diálise
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Liofilização
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Cromatografia= separação de componentes de uma mistura com base nas
diferenças de adsorção à uma matriz insolúvel
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Cromatografia em coluna
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Cromatografia de GEL FILTRAÇÃO
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Cromatografia de GEL FILTRAÇÃO
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Cromatografia de GEL FILTRAÇÃO
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Cromatografia de GEL FILTRAÇÃO
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Gel filtração : Sephadex - Agarose
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Gel filtração : colunas
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GEL FILTRAÇÃO
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GEL FILTRAÇÃO- calibraçãoGlucagon
Citocromo c
Ribonucleasa
Seroalbúmina
IgGTiroglobulina
Volumen
Peso molecular
280
240
200
160
120
80
40
103
104
105
106
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GEL FILTRAÇÃO
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Purificação de proteínasCromatografia
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Equipamentos, soluções e resinas• Coluna de vidro com filtro
• Coletor de frações
• Bomba peristáltica
• Soluções tampão de pH
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Espectrofotômetros
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GEL FILTRAÇÃO
• Baseia-se no peso molecular das proteínas da amostra
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GEL FILTRAÇÃO
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TROCA-IÔNICA
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O
OH
HOCH2
OHH
H
HOO
O
OH
HOCH2
OHO
H
H
H
H
nCelulosa
O
O
H
H
OHCH2
O
H
O
O
OH
OH
HOCH2
HO
H
H
n
Agarosa
O
OH
CH2
OH
H
HOH
O
OH
OH
H
H
CH2O
H
O
H
H
OH
H
O
n
Dextrano
SUPORTES CROMATOGRÁFICOS
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CH2 COO-
Carboximetil (CM)
CH2 CH2 S
O
O
O-
Sulfoetil (SE)
CH2 CH2 CH2 S
O
O
O-
Sulfopropil (SP)
O P
OO-
O-
Fosfo (P)
TROCADOR CATIÔNICO
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CH2 CH2 NH+CH2
CH2 CH3
CH3
Dietilaminoetil (DEAE)
CH2 CH2 N+ CH2 CH3CH2CH3
CH2
CH3
Trietilaminoetil (TEAE)
CH2 CH2 N+ CH2CH2CH3
CH2
CH3
CHOH CH3
Dietil 2-hidroxipropil aminoetil (QAE)
TROCADOR ANIÔNICO
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+
+++
+++++
---
-
----
--
+
+++
+++++
-
--
--
--
---
- -- -
-
---
-
+
+++
+++++
-
-
--
--
---
-
-
---
-
- -
--
-
Proteínas ligadas
ao trocadoriônico
Baixas [SAL]Desprendem-se
as proteínas menoseletronegativas
Maiores [SAL]Desprendem-se
as proteínas maiseletronegativas
TROCA-IÔNICA
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Cromatografia de troca- iônica
Bomba peristáltica
Generadorde gradiente
Columna
Colector defracciones
Resina deintercambioiónico
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Baseia-se na AFINIDADE pelo suporte acoplado à resina
AFINIDADE
1 2 3
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Cromatografia de afinidade
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ELETROFORESE EM GEL DE
POLIACRILAMIDA COM SDS
SDS-PAGE
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+
-
ELETROFORESE EM GEL DE POLIACRILAMIDA COM SDS
SDS-PAGE
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Coloração com Coomassie Blue
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Coloração por prata
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