Protein purification and Analysis

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• 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
transfer supernatant
transfer supernatant
transfer supernatant
1000 g
10,000 g
100,000 g
Pellet microsomal Fraction (ER,
golgi, lysosomes, peroxisomes)
Pellet unbroken cells nuclei chloroplast
Pellet mitochondria
Super. Cytosol, Soluble enzymes
tissue homogenate
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Chromatography
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Gel Permeation Chromatography
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Ion-exchange Chromatography
low salt buffer
high salt buffer
- - -
- - -
- - -
Cl-
Cl-
Cl-
- - -
Cl-
- - -
Cl-
Cl-
- - -
- - -
- - -
- - -
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Affinity Chromatography
Add excess ligand
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SDS poly acrylamide electrophoresis (PAGE)
SDS H3C-(CH2)11-CH2-OSO3-
SDS denatures protein coats w/ negative charge
Used to determine protein MW And purity of
protein prep
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Isoelectric Focusing
-
-
pH 9
Decreasing pH
Decreasing pH
pH 3


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2-D Electrophoresis
Decreasing pH
-
SDS-PAGE
Decreasing MW

Decreasing pH
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Amino Acid Analysis

1. Acid hydrolyze protein
2. Treat with phenylisothiocyanate (PICT)
3. Separate derivatized AAs by HPLC

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Protein Sequencing (Edman Degradation)
1)
2)
Repeat
Trifluoroacetic acid

3)
Can sequence in 30 to 60 AAs 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 fragments
• Sequence individual fragements and piece together
  sequence

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Peptide mapping exercise
Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-G
ln-Asp
Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Ty
r-Met-Cys-Lys
Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys
Phe Ala-Glu-Gln-Asp
CNBr Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys
-Phe-Ala-Glu-Gln-Asp