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    Biochemistry LN06

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    Rahaf Al-muhtaseb
    This document provides an overview of techniques for extracting and purifying proteins from cells. It discusses homogenization to break open cells, followed by differential centrifugation to separate subcellular components. Crude purification using ammonium sulfate precipitation separates proteins based on solubility. Further purification steps like dialysis and diafiltration remove salts and other reagents to isolate the target protein while stabilizing its structure and activity. Maintaining proper pH, temperature, and adding protease inhibitors are important to preserve protein function during purification. The goal is to recover as much of the target protein as possible at increasing purity levels across the multiple separation techniques.

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    Biochemistry LN06

    Uploaded by

    Rahaf Al-muhtaseb
    23 views10 pages
    This document provides an overview of techniques for extracting and purifying proteins from cells. It discusses homogenization to break open cells, followed by differential centrifugation to separate subcellular components. Crude purification using ammonium sulfate precipitation separates proteins based on solubility. Further purification steps like dialysis and diafiltration remove salts and other reagents to isolate the target protein while stabilizing its structure and activity. Maintaining proper pH, temperature, and adding protease inhibitors are important to preserve protein function during purification. The goal is to recover as much of the target protein as possible at increasing purity levels across the multiple separation techniques.

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    Biochemistry_LN06

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    This document provides an overview of techniques for extracting and purifying proteins from cells. It discusses homogenization to break open cells, followed by differential centrifugation to separate subcellular components. Crude purification using ammonium sulfate precipitation separates proteins based on solubility. Further purification steps like dialysis and diafiltration remove salts and other reagents to isolate the target protein while stabilizing its structure and activity. Maintaining proper pH, temperature, and adding protease inhibitors are important to preserve protein function during purification. The goal is to recover as much of the target protein as possible at increasing purity levels across the multiple separation techniques.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    23 views10 pages

    Biochemistry LN06

    Uploaded by

    Rahaf Al-muhtaseb
    This document provides an overview of techniques for extracting and purifying proteins from cells. It discusses homogenization to break open cells, followed by differential centrifugation to separate subcellular components. Crude purification using ammonium sulfate precipitation separates proteins based on solubility. Further purification steps like dialysis and diafiltration remove salts and other reagents to isolate the target protein while stabilizing its structure and activity. Maintaining proper pH, temperature, and adding protease inhibitors are important to preserve protein function during purification. The goal is to recover as much of the target protein as possible at increasing purity levels across the multiple separation techniques.

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    Biochemistry

    Dr. Tareq Alhindi

    Lecture Notes (06)


    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells

    Many different proteins exist in a single cell.


    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells

    A detailed study of the properties of any one protein


    requires a homogeneous sample consisting of only one
    kind of molecule.

    Separation techniques focus on:


    - Size
    - Charge
    - Polarity
    Percent recovery: a measurement of the amount of an enzyme
    recovered at each step of a purification experiment.
    As the purification steps are followed, we make a table
    of the recovery and purity of the protein to gauge our This number usually drops steadily during the purification.
    success.
    Specific activity: compares the purity of the protein at each
    step, and this value should go up if the purification is
    successful.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells

    Before the real purification steps can begin, the protein The squeezing of the homogenate
    must be released from the cells and subcellular around the plunger breaks open cells,
    organelles. but it leaves many of the organelles
    The first step, called homogenization, involves breaking intact.
    open the cells. Sonication, involves using
    sound waves to break open the
    Mechanical techniques cells.

    Grinding the tissue in a blender with a suitable buffer. Cells can also be ruptured by
    cycles of freezing and
    This process also breaks many of the subcellular thawing.
    organelles, such as mitochondria, peroxisomes, and
    endoplasmic reticulum. Releasing soluble proteins. Vortex with glass or metal beads.
    Potter–Elvehjem homogenizer, is similar but more
    gentle.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells

    If the protein of interest is solidly attached to a Proteolysis can be a problem whenever cells are
    membrane, or when cells have a very tough cell wall; manipulated; therefore, protease inhibitors should be added
    detergents may have to be added to detach the proteins, to all samples undergoing lysis.
    or degrade the cell wall.

    Reagent-based techniques

    Lysozyme can be used to digest the polysaccharide


    component of yeast and bacterial cell walls.

    DNase and RNase are added to digest nucleic acid


    material. Viscosity of a sample typically increases during
    lysis due to the release of nucleic acid material.

    Nuclease treatment is not required for sonicated


    material since sonication shears chromosomes.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells

    Reagent-based methods characteristics Mechanical methods characteristics


    Pros Pros
    - It is a rapid, gentle, efficient, and reproducible - It can be applied on industrial scale.
    method, and leads to a high protein yield. - Hazardous chemicals are not required
    - It can be used to extract total protein or subcellular - Easier to use will downstream processes.
    fractions or organelles from various sample types. Cons
    - It works by disrupting the lipid membrane and/or - It may require expensive equipment.
    cell wall. - It may be cumbersome to use, and time consuming.
    Cons - Reproducibility may vary.
    - Some components may need to be removed for - Mechanical methods are generally not compatible with
    downstream analysis. high-throughput and small volumes.
    - High concentrations of salts and detergents are not - Protein denaturation and aggregation can occur.
    compatible with protein assays and mass - Cells disrupt at different times, so subcellular
    spectrometry. components may be subjected to ongoing disruptive
    forces.
    Protein Purification and
    Characterization Techniques
    Extracting Pure Proteins from Cells

    After the cells are homogenized, they are subjected to


    differential centrifugation.

    Spinning the sample at 600 times the force of gravity


    (600 x g) results in a pellet of unbroken cells and nuclei.

    If the protein of interest is not found in the nuclei, this


    precipitate is discarded.

    The supernatant can then be centrifuged at higher


    speed, such as 15,000 x g, to bring down the
    mitochondria.
    Centrifugation at 100,000 x g brings down the
    microsomal fraction, consisting of ribosomes and
    membrane fragments.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells
    With less water available to hydrate the proteins, they begin to
    After the proteins are solubilized, they are often interact with each other through hydrophobic bonds and
    subjected to a crude purification based on solubility. coalesce. At a defined amount of ammonium sulfate, a
    precipitate that contains contaminating proteins forms.
    Ammonium sulfate is the most common reagent to
    use at this step, and this procedure is referred to as These proteins are
    salting out. centrifuged down and
    discarded. Then more salt is
    Proteins have varying solubilities in polar and ionic added, and a different set of
    compounds. Proteins remain soluble because of proteins, which usually
    their interactions with water. contains the protein of
    interest, precipitates. This
    When ammonium sulfate is added to a protein precipitate is collected by
    solution, some of the water is taken away from the centrifugation, resuspended
    protein to make ion–dipole bonds with the salts. in a suitable buffer, and saved
    for further purification steps.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells
    Stabilizing proteins in solution

    The proteins must be stabilized to maintain its Natural proteases that can digest the target proteins in a
    bioactivity. preparation are another threat. Protease inhibitors and
    antimicrobials can be added to prevent the protein
    Although it is essential that the protein be purified, molecules from being dismantled but must be removed
    it is equally important that the protein maintain its later, as must any additive used in the purification process.
    biological activity.

    Maintaining a low temperature is crucial to Another potential problem is mechanical destruction by


    protecting proteins, so most purifications must occur foaming or shearing of the proteins into useless fragments.
    at low temperatures. Again, additives can help prevent foaming and shearing from
    destroying the protein, but the additives must be removed
    Maintaining the proper pH for the activity of a protein later.
    is also important, and most active proteins are
    suspended in buffering agents to preserve maximal
    function.
    Protein Purification and Characterization Techniques
    Extracting Pure Proteins from Cells
    Diafiltration and dialysis are filtration methods that rely Dialysis depends on the ability of some molecules to pass
    on the chemical concept of equilibrium, the migration through semipermeable membranes while others are halted
    of dissolved substances from areas of higher or slowed because of their size.
    concentration to areas of lower concentration.
    Dialysis is often required to remove the smaller salts, solvents,
    and other additives used earlier in the purification. The salts
    are then replaced with buffering agents that help stabilize the
    proteins during the remainder of the process.
    Diafiltration adds a filtering component to dialysis.

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