chapter 3(part2) - protein purification and analysis
DESCRIPTION
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Chapter 3 (part 2)
Protein purification and Analysis
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Why purify proteins?
• Pure proteins are required to study enzyme function
• Pure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy)
• Pure proteins are required to obtain amino acid sequence
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Steps in protein purification
• Develop assay • Choose source of protein• Prepare tissue extract
– cell disruption– subcellular fractionation
• Protein fractionation (several steps)
• Determination of purity
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Differential Centrifugation
tissuehomogenate
1000 g
Pelletunbroken cells
nucleichloroplast
transfer supernatant
transfer supernatant
transfer supernatant
10,000 g 100,000 g
Pelletmitochondria
Pelletmicrosomal
Fraction(ER, golgi,lysosomes,
peroxisomes)
Super.Cytosol,Soluble
enzymes
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Chromatography
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Gel Permeation Chromatography
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+++
++
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+++++
++
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+++++
++
+++
++
+++
++
+++
++
- - -- - -- - -
++++++
++++++
++++++
+++
++
+++
+++++
++
+++
+++++
++
+++
++
+++
++
+++
++
- - -- - -- - -
Cl-Cl-
Cl-Cl-Cl-
Cl-
+++
++
+++
+++++
++
+++
+++++
++
+++
++
+++
++
+++
++
- - -
- - -- - -
++++++++++++++++++
low salt buffer high salt buffer
Ion-exchange Chromatography
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Affinity ChromatographyAdd excess ligand
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SDS poly acrylamide electrophoresis (PAGE)SDS = H3C-(CH2)10-CH2-OSO3
-
-- - -
--
- ---
-
-
----
SDS – denatures proteincoats w/ negative charge
Used to determine protein MWAnd purity of protein prep
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Isoelectric FocusingD
ecr
easi
ng
pH
+
-
Decr
easi
ng
pH
+
-pH 9
pH 3
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2-D ElectrophoresisD
ecr
easi
ng
MW
small
largeDecreasing pH
+
-SDS-PAGE
Decreasing pH
Decre
asin
g M
W
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Amino Acid Analysis
H3N C
H
C
O
R
O-N C S N
C
S
HN
CC
H
O
R
1) Acid hydrolyze protein2) Treat with phenylisothiocyanate (PICT)
3) Separate derivatized AA’s by HPLC
+
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Protein Sequencing (Edman Degradation)
N C S H3N C
H
C
O
R
NH C
H
C
O
R
X
N C
S
HN C
H
C
O
R
NH C
H
C
O
R
X
+
Trifluoroacetic acid
N
C
S
HN
CC
H
O
R
2HN C
H
C
O
R
X
1)
2)
3)
Rep
eat
Can sequence in 30 to 60 AA’s from N-terminus
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Generate Proteolytic Fragments
Endopeptidases
•Typsin cleaves at COOH end of Lys and Arg•Chymotrypsin cleaves at COOH end of Phe, Tyr, Trp
Chemical Cleavages
•Cyanogen Bromide cleaves at COOH end of Met
Generate overlapping fragmentsSequence individual fragments and piece together sequence
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Peptide mapping exerciseMet-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp
TrypsinMet-Ala-ArgPhe-Ala-Glu-Gln-AspGly-Glu-Tyr-Met-Cys-Lys
ChymotrysinMet-Ala-Arg- Gly-Glu-TyrMet-Cys-Lys –PheAla-Glu-Gln-Asp
CNBrMetAla-Arg-Gly-Glu-Tyr-MetCys-Lys-Phe-Ala-Glu-Gln-Asp
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Proteomic Analysis
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Matrix Assisted Laser Desorption Ionization Time of Flight
(MALDI-TOF)