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    Protein Isolation From Whole Blood

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    Shadia Heyari
    This document describes the process of isolating total proteins from white blood cells in sheep blood. The procedure involves lysing red blood cells with NH4Cl solution, centrifuging to separate blood components by density, resuspending the white blood cell pellet in PBS, centrifuging again, and extracting proteins using a buffer containing SDS, Tris, NaF, DTT, and EGTA before boiling and storing on ice. The buffer components work to denature and extract proteins while preserving phosphorylation states. Proteins are composed of amino acids linked by peptide bonds and folded into structures like alpha helices and beta sheets that determine function.

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    Protein Isolation From Whole Blood

    Uploaded by

    Shadia Heyari
    80 views8 pages
    This document describes the process of isolating total proteins from white blood cells in sheep blood. The procedure involves lysing red blood cells with NH4Cl solution, centrifuging to separate blood components by density, resuspending the white blood cell pellet in PBS, centrifuging again, and extracting proteins using a buffer containing SDS, Tris, NaF, DTT, and EGTA before boiling and storing on ice. The buffer components work to denature and extract proteins while preserving phosphorylation states. Proteins are composed of amino acids linked by peptide bonds and folded into structures like alpha helices and beta sheets that determine function.

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    Protein isolation from Whole Blood

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    This document describes the process of isolating total proteins from white blood cells in sheep blood. The procedure involves lysing red blood cells with NH4Cl solution, centrifuging to separate blood components by density, resuspending the white blood cell pellet in PBS, centrifuging again, and extracting proteins using a buffer containing SDS, Tris, NaF, DTT, and EGTA before boiling and storing on ice. The buffer components work to denature and extract proteins while preserving phosphorylation states. Proteins are composed of amino acids linked by peptide bonds and folded into structures like alpha helices and beta sheets that determine function.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    80 views8 pages

    Protein Isolation From Whole Blood

    Uploaded by

    Shadia Heyari
    This document describes the process of isolating total proteins from white blood cells in sheep blood. The procedure involves lysing red blood cells with NH4Cl solution, centrifuging to separate blood components by density, resuspending the white blood cell pellet in PBS, centrifuging again, and extracting proteins using a buffer containing SDS, Tris, NaF, DTT, and EGTA before boiling and storing on ice. The buffer components work to denature and extract proteins while preserving phosphorylation states. Proteins are composed of amino acids linked by peptide bonds and folded into structures like alpha helices and beta sheets that determine function.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 8

    Isolating total proteins from whole blood

    In this experiment we will isolate protein from White Blood Cells (WBC) of
    sheep, as human blood is biohazard material!

    • Procedure:

    1. 3 ml blood, Add 9.0ml of RBC (Red Blood Cells) lysis


    solution (0.84% NH4Cl) , Invert the tube 5-6 times to mix.
    NH4Cl is a salt that cause bursting of the cell releasing its
    content due to osmosis effect

    2. Incubate the mixture for 10 minutes at room temperature


    (invert 2-3 times during the incubation) to lyse the red blood
    cells.

    3. Centrifuge at 4000 RPM for 10 minutes at room


    temperature; in this step, you separate the component
    of the blood according to their densities, leading to have
    heavier component on the bottom and the lighter ones Centrifuge
    above.
    4. Discard supernatant by decantation, Keep the visible white pellet:
    the pellet here consist of intact WBC that is heavier in density, while the
    supernatant is composed of lighter substances, such as lysed RBC, NH4Cl and
    serum.

    All the previous steps are done at Room Temperature , further steps will
    be performed in an ice box, why?

    - Because in the later steps you will extract protein from cells to the
    surrounding that is full of proteases leading to destruction and
    denaturation of them, in ice proteases will be inhibited.

    5. Add 500 µl of cold Phosphate Buffer Saline (PBS) and pipette the cells
    vigorously to resuspend the pellet (10-15 seconds).

    6. Move the suspension to a 1.5 ml microcentrifuge tube and centrifuge at


    maximum speed for 1 minute at 4oC.
    7. discard the supernatant, Keep the visible white pellet.
    8. Add 100-150 μl of the extraction buffer. Vortex or pipette thoroughly
    until all clumps disappear and your sample becomes viscous.
    9. Boil the sample for 3-5 minutes then keep on ice for the next experiment.
    • Why boiling?
    To completely denature the proteins to homogenate the sample and to
    make it less gummy by melting the DNA in the sample.

    To know what is a micropipette and when we use it watch:


    https://www.youtube.com/watch?v=QGX490kuKjg&feature=share&fbclid=Iw
    AR1QIXPBY1N-nha7Rao7hr5s9PW57xTI9Qk5Qv8PT7ltZYmdAgk1zbljMi0

    To know about centrifuge device and its purpose, watch:


    https://www.youtube.com/watch?v=F3788O7jy2I&feature=share&fbclid=IwA
    R071i5bGU1BIwHAQ2wCUBDKBJqfdtj52-HvOS5-RbeclLhZEKjww5ewn6E

    To learn about vortex mixer that is used for vigorous mixing, watch:
    https://www.youtube.com/watch?v=aGbogxCyTq0
    Extraction buffer components:
    • 10% SDS: anionic detergent , denatures proteins by disturbing the non
    covalent forces, also gives a net negative charge for the proteins.
    • 1M Tris buffer pH 7.5: maintain the pH constant and protect the proteins.
    • 1M NaF: inhibitor for protein phosphoseryl and phosphothreonyl
    phosphatases (PSPs), to preserve the proteins phosphorylation state in the
    cells.
    • 1M DTT: reduces the disulfide bonds of proteins, To prevent intramolecular
    disulfide bonds from forming cysteine residues of proteins.
    • 0.1M EGTA: chelating agent and inhibitor
    • distilled water
    • Protein structure:
    Proteins are the end products of the decoding process that starts with
    the information in cellular DNA. As workhorses of the cell, proteins
    compose structural and motor elements in the cell. In fact, each gene in
    cellular DNA contains the code for a unique protein structure. Not only
    are these proteins assembled with different amino acid sequences, but
    they also are held together by different bonds and folded into a variety
    of three-dimensional structures. The folded shape, or conformation,
    depends directly on the linear amino acid sequence of the protein.

    • What Are Proteins Made Of?


    The building blocks of proteins are amino acids, which are small organic
    molecules that consist of an alpha (central) carbon atom linked to an
    amino group, a carboxyl group, a hydrogen atom, and a variable
    component called a side chain.
    • Within a protein, multiple amino acids are linked together
    by peptide bonds, The linear sequence of amino acids within a
    protein is considered the primary structure of the protein.
    • The primary structure of a protein — its amino acid sequence
    — drives the folding and intramolecular bonding of the linear
    amino acid chain, which ultimately determines the protein's
    unique three-dimensional shape. Hydrogen bonding between
    amino groups and carboxyl groups in neighboring regions of
    the protein chain sometimes causes certain patterns of folding
    to occur. Known as alpha helices and beta sheets, these stable
    folding patterns make up the secondary structure of a protein.
    • The ensemble of formations and folds in a single linear chain
    of amino acids — sometimes called a polypeptide —
    constitutes the tertiary structure of a protein. Finally,
    the quaternary structure of a protein refers to those
    macromolecules with multiple polypeptide chains or subunits.

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