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  • Methods For Protein Analysis Wiiiiiiiii

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    Angela Sietereales Ramos
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    This document outlines the steps for determining the amino acid composition and sequence of a protein. It describes: 1) Hydrolyzing the protein into individual amino acids using acid or base hydrolysis. 2) Separating and quantifying the amino acids using high-performance liquid chromatography after derivatization. 3) Cleaving disulfide bonds and determining the N-terminal residue, often using Edman degradation. 4) Fragmenting the protein using enzymatic or chemical cleavage at specific amino acid residues. 5) Determining the amino acid sequence, typically using Edman degradation and the overlapping technique.

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    BIO 101 Report PPT

    Original Title

    Methods for Protein Analysis Wiiiiiiiii

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    This document outlines the steps for determining the amino acid composition and sequence of a protein. It describes: 1) Hydrolyzing the protein into individual amino acids using acid or base hydrolysis. 2) Separating and quantifying the amino acids using high-performance liquid chromatography after derivatization. 3) Cleaving disulfide bonds and determining the N-terminal residue, often using Edman degradation. 4) Fragmenting the protein using enzymatic or chemical cleavage at specific amino acid residues. 5) Determining the amino acid sequence, typically using Edman degradation and the overlapping technique.

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    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
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    7 views12 pages

    Methods For Protein Analysis Wiiiiiiiii

    Uploaded by

    Angela Sietereales Ramos
    This document outlines the steps for determining the amino acid composition and sequence of a protein. It describes: 1) Hydrolyzing the protein into individual amino acids using acid or base hydrolysis. 2) Separating and quantifying the amino acids using high-performance liquid chromatography after derivatization. 3) Cleaving disulfide bonds and determining the N-terminal residue, often using Edman degradation. 4) Fragmenting the protein using enzymatic or chemical cleavage at specific amino acid residues. 5) Determining the amino acid sequence, typically using Edman degradation and the overlapping technique.

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    of 12

    METHODS

    FOR PROTEIN
    ANALYSIS
    Group 2
    RAMOS, ROYO, SAGARIO, SAEZ,
    SALVATIERRA, SAMBO, SARMIENTO

    Steps in Amino Acid


    Composition Determination
    1. Hydrolysis - breaking down of
    polypeptide chain into its component amino
    acids
    Acid hydrolysis
    - (6M HCl, 100C, 24 hours)
    - Has varied effects on different amino
    acids
    Base hydrolysis
    -(4N NaOH, 100C, 10 hours)

    2. High Pressure Liquid


    Chromatography (HPLC)

    AKA amino acid composition chromatogram


    Separation and quantification of free amino
    acids present

    a) Treatment of hydrosylate with


    phenylisothiocyanate
    (PITC) phenylcarbamoyl (PTC) amino acid
    derivatives
    b) PTC-AA subjected to HPLC
    c) Eluted PTC-AA detected and concentration
    is
    determined by measuring absorbance
    at 254 nm (peak absorbance of PTC-AA moeity)

    Steps in Amino acid


    Sequencing
    1. Purify the polypeptide
    2. Cleave disulfide bonds
    * Performic acid, OR

    * dithiothreitol and iodoacetate

    Reduction by
    dithiothreitol

    Acetylation by
    iodoacetate

    3.

    Determination of

    N-TERMINAL RESIDUE
    METHOD

    Sangers Method
    Edman degradation
    Enzyme

    LABELLING COMPOUND

    2,4-dinitriflourobenzene
    (DNFB)
    Phenyl isothiocyanate
    Aminopeptidase

    Dansyl chloride

    10dimethylaminonapthalene5-sulfonyl chloride

    Dabsyl chloride

    4dimethylaminoazobenzene
    -4-sulfonyl chloride

    EDMAN
    DEGRADATION
    PRODEDURE

    Labels and removes


    N-terminal residue
    only; leaves the rest
    of the chain intact
    up to 50 AA
    For shorter peptides,
    only this step is done.

    4. Fragment the polypeptide by


    internal cleavage.
    Enzyme/
    Chemical
    Cyanogen
    bromide
    Trypsin

    Side

    Carboxyl
    side
    Carboxyl
    side
    Chymotrypsin
    Carboxyl
    side
    Elastase
    Carboxyl
    side
    Thermolysin Amino side

    Amino Acids
    Cleaved
    Met
    Arg, Lys
    Tyr, Phe, Trp, Met,
    Leu
    Val, Thr
    Ile, Leu, Val, Phe,
    Trp, Tyr

    5. Determine amino acid


    sequence
    Edman Degradation

    6. Perform the overlapping


    technique

    References
    Berg J.M., Tymoczko J.L., and Stryer L. 2002.
    Biochemistry. 5th ed.
    New York:W. H. Freeman.
    Horton, H.R., Moran, L.A., Ochs, R.S., Rawn, J.D.,
    and Scrimgeour, K.G., 1993. Principles of
    Biochemistry. USA: Neil Patterson
    Publishers/Prentice-Hall, Inc.
    Lehninger, A.L., Nelson, D.M., and Cox, M.M. 1993.
    Principles of Biochemistry. 2nd ed. New York: Worth.

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