methods in protein analysis biochemistry
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Methods in Protein Biochemistry
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Gel Electrophoresis
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Polyampholyte Character of aTetrapeptide and Isoelectric Points
Isoelectric Point (pI) , pH at
which molecule has net zerocharge, determined usingcomputer program for knownsequence or empirically (byisoelectric focusing).
Group pKaa -NH 3+ 9.7 Glu g-COOH 4.2Lys e -NH 3+ 10.0a -COOH 2.2
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Salting Out: Ammonium SulfatePrecipitation in Protein Fractionation
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Centrifugation
Centrifugation Methods
Differential (Pelletting) simple methodfor pelleting large particles using fixed-angle rotor (pellet at bottom of tube vs.supernatant solution above)
Zonal ultracentrifugation ( e.g. , sucrose-gradient) swinging-bucket rotor
Equilibrium-density gradientultracentrifugation ( e.g. , CsCl) swinging-bucket or fixed-angle rotor
Low-speed, high-speed, orultracentrifugation: differentspin speeds and g forces
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Zonal Centrifugation: Sucrose-GradientPreparative Ultracentrifugation
Separates by sedimentation coefficient (determined
by size and shape of solutes)
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Sucrose-Gradient PreparativeUltracentrifugation
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Equilibrium Density GradientUltracentrifugation
Used in Meselsen-Stahl experiment. Separates based on densities of solutes. Does not require premade gradient. Pour dense solution of rapidly diffusing substance in
tube (usually CsCl). Density gradient forms during centrifugation (self -
generating gradient).
Solutes migrate according to their buoyant density(where density of solute = density of CsCl solution).
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Column Chromatography
Flow-through Eluate
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Different Types of Chromatography
Gel filtration/size exclusion/molecular sieve - separates by size(molecular weight) of proteins
Ion exchange (cation exchange and anion exchange) -
separates by surface charge on proteins Cation exchange: separates based on positive charges of
solutes/proteins, matrix is negatively charged Anion exchange: separates based on negative charges of
solutes/proteins, matrix is positively charged
Hydrophobic interaction - separates by hydrophobicity ofproteins Affinity - separates by some unique binding characteristic of
protein of interest for affinity matrix in column
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Ion-Exchange Chromatography
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Gel Filtration Chromatography
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Affinity Chromatography
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Cleavage of Polypeptides for Analysis
Strong acid ( e.g. , 6 M HCl) - not sequence specific
Sequence-specific proteolytic enzymes (proteases) Sequence-specific chemical cleavage ( e.g. ,
cyanogen bromide cleavage at methionine residues)
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Protease Specificities
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Cyanogen Bromide Cleavage atMethionine Residues
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Protein Sequencing: Edman Degradation
PTH = phenylthiohydantion
F3CCOOH = trifluoroacetic acid
PTC = phenylthiocarbamyl
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Identification of N-Terminal Residue
Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildlyacidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase).
NO 2
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Separation of Amino Acids by HPLC
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Protein Identification by MassSpectrometry
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Protein Identification by MassSpectrometry
Two main approaches:
1. Peptide mass fingerprinting: Proteolytic digestion of protein,then determination m /z of peptides by MS ( e.g. , MALDI-TOFor ESI- TOF), search fingerprint against database. Successof ID depends on quality/ completeness of database forspecific proteome.
2. Tandem MS (MS/MS e.g. , nanoLC-ESI-MS/MS):Proteolytic digestion of protein, separation and determinationof m /z of each (MS-1), then determination of collision-induceddissociation fragment spectrum for each peptide (MS-2).Gives context/sequence-dependent information, so more of ado novo sequencing method.
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Locating Disulfide Bonds
O
O -I
iodoacetate
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Determing Primary Structure of an EntireProtein
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Reactions in Solid-Phase PeptideSynthesis
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