Last Lecture: Protein PurificationCharacterization

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Last Lecture Protein Purification/Characterizati
on Today Purification/Characterization,
Protein 1 Structure
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Affinity Chromatography
Antibody
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Affinity Chromatography
Tagged Proteins GST-Tag
Glutathione S-Transferase
Glutatione (GSH)
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Affinity Chromatography
Tagged Proteins His6-Tag Ni2-chelation
Imidazole
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Electrophoresis
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SDS-PAGE
PAGE - polyacrylamide gel electrophoresis
SDS - sodium dodecyl sulfate
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SDS-PAGE
molecular weight markers
_

Coomassie blue stain
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SDS-PAGE
-
200 kD
14 kD

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LYMPHOMA SWISS-2DPAGE map
Source http//www.expasy.ch/ch2d/
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Protein Structure
Primary (1) amino acid sequence, disulfide
bridges
Secondary (2) regular structure
- alpha helix (a)
-beta sheet (b)
Tertiary (3) fold of structure
Quaternary (4) interaction of sub-units
-higher order structures
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Protein Folding
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Folding/Unfolding
Folding small energetic advantage
-chaperones
Unfolding temperature, denaturants (SDS, urea,
guanidine) reducing agents
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Peptide Mapping/Sequencing Reagents for Specific
Cleavages
1. Enzymes Trypsin (Lys, Arg) Chymotrypsin
(Phe, Tyr, Trp) Elastase (Ala, Ser, Val,
Gly) Lys-C (Lys),
Trypsin
WDGK TL
ie. WDGKTL
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Peptide Mapping/Sequencing Reagents for Specific
Cleavages
2. Cyanogen Bromide Br-CN, cleavage after Met
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Amino Acid Analysis
- total hydrolysis 6 N HCl 24 hrs - release
of amino acids
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Amino Acid Analysis
sulfonated polystyrene
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Ninhydrin Reaction
purple-blue (lmax 570 nm)
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Peptide Sequencing
http//www.ncbi.nlm.nih.gov/BLAST/
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Peptide Sequencing -Chemistry
Edman Degradation
Phenylisothiocyanate (PTC)
PTC
PTC derivative
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