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    Virtual Lab 2

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    JV Gamo
    The process of protein purification involves three main stages: capture, intermediate purification, and polishing. In the capture stage, the target protein is isolated and concentrated using techniques like 2D gel electrophoresis and immunoblotting. Next, intermediate purification eliminates most impurities like nucleic acids and other proteins, using gel filtration. Finally, polishing removes remaining impurities by assaying enzyme activity and pooling active fractions, then using additional techniques like ion exchange chromatography. The document describes each stage of purifying a target protein, monitoring enrichment levels and isolating fractions with enzyme activity at each step.

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    Virtual Lab 2

    Uploaded by

    JV Gamo
    209 views11 pages
    The process of protein purification involves three main stages: capture, intermediate purification, and polishing. In the capture stage, the target protein is isolated and concentrated using techniques like 2D gel electrophoresis and immunoblotting. Next, intermediate purification eliminates most impurities like nucleic acids and other proteins, using gel filtration. Finally, polishing removes remaining impurities by assaying enzyme activity and pooling active fractions, then using additional techniques like ion exchange chromatography. The document describes each stage of purifying a target protein, monitoring enrichment levels and isolating fractions with enzyme activity at each step.

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    The process of protein purification involves three main stages: capture, intermediate purification, and polishing. In the capture stage, the target protein is isolated and concentrated using techniques like 2D gel electrophoresis and immunoblotting. Next, intermediate purification eliminates most impurities like nucleic acids and other proteins, using gel filtration. Finally, polishing removes remaining impurities by assaying enzyme activity and pooling active fractions, then using additional techniques like ion exchange chromatography. The document describes each stage of purifying a target protein, monitoring enrichment levels and isolating fractions with enzyme activity at each step.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    209 views11 pages

    Virtual Lab 2

    Uploaded by

    JV Gamo
    The process of protein purification involves three main stages: capture, intermediate purification, and polishing. In the capture stage, the target protein is isolated and concentrated using techniques like 2D gel electrophoresis and immunoblotting. Next, intermediate purification eliminates most impurities like nucleic acids and other proteins, using gel filtration. Finally, polishing removes remaining impurities by assaying enzyme activity and pooling active fractions, then using additional techniques like ion exchange chromatography. The document describes each stage of purifying a target protein, monitoring enrichment levels and isolating fractions with enzyme activity at each step.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 11

    Virtual Lab: Protein

    Purification
    JV Gamo | CH152 - B

    The process of protein purification may be divided into three main parts: capture, intermediate
    purification, and polishing. (MCB Harvard University). In the capture stage, the target protein is
    isolated and concentrated. The process of 2D page and immunoblotting is part of this stage. Next is
    the intermediate purification stage, where most of the impurities, such as nucleic acid and other
    proteins, are eliminated. The process of gel filtration is part of this stage. Lastly, the polishing
    stage is where the rest of the remaining impurities are discarded. The process of assaying enzyme
    activity and subsequently pooling fractions of the sample based on the assay, are included in this
    final stage.
    Seen here in the image above, the enrichment value is a mere 1.0, by the end of the protein
    purification process, the enrichment level will have been raised by a significant amount.

    The first process done is the two-dimensional gel electrophoresis or 2D page,


    which is commonly used for proteins. Herein, protein mixtures are separated
    by two properties in two dimensions on 2-D gels. The two properties
    identified isoelectric point in the above example are isoelectric point (x-axis)
    and protein mass (y-axis).

    The above picture shows immunoblotting, which is a technique used to


    analyze or identify proteins in a mixture. It involves separation by
    electrophoresis and subsequent staining with antibodies to isolate the protein
    in question. This shows at which isoelectric point value and protein mass
    value the target protein is located (pH 8.0 and 55,000 Mr, respectively)

    Now, we have the task to choose the most appropriate gel matrix to use for
    the gel filtration. Ultragel AcA 34 has the only appropriate fractionation
    range of 20,000 400,000 molecular weight, which included 55, 000, the MW
    of our protein, within its range.

    In the graph shown above, the red portion is the only fraction of the sample
    wherein enzymatic activity is observed. This observation is possible due to
    the enzymatic assay procedure, which is for the purpose of measuring
    enzyme activity.
    Portions 75-90 were then subsequently pooled and isolated because they
    were the only fractions that exhibited enzyme activity.

    After, the gel filtration procedure, the enrichment of the protein is now up to
    4.7, from 1.0. This means that some of the impurities have now been
    eliminated. But lets see if we can do another procedure to eliminate the rest
    of the impurities.

    Here is where another separation process


    called
    ion exchange
    chromatography comes in. Separation through this process is based upon
    the reversible adsorption of charged molecules to the inactive ion exchange
    groups of the opposite charge. (MCB Harvard University). Hereafter, the some
    or most, if not all, of the remaining impurities can be discarded.

    In the graph shown above, the red portion, again, is the only fraction of the
    sample wherein enzymatic activity is observed. This observation is possible
    due to the enzymatic assay procedure.
    The fractions within the red curve (i.e. 60-120), are then spooled and isolated
    because, again, they were the only fractions that exhibited enzyme activity.

    After the ion exchange chromatography process, the enrichment value is now
    up to 51.1 from 4.7, which means more impurities have been eliminated after
    this step. There is no particular indicator as to how purified the protein has
    been, but the increase of the enrichment level is a telltale sign of purification,
    per se.

    SOURCES
    http://www.agbooth.com/pp_tut_onlin
    e/Protein4.html
    http://labs.mcb.harvard.edu/Gaudet/
    Resources_Files/GEHealthcare_chrom
    atography/Don't
    %20move/18113229AB.pdf

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