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    Protein Purification Questions

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    Hey I am did a multiweek experiment doing a protein purification of Glutamate-Oxaloacetate Transaminase (GOT). I have a few questions about my experiment.

    1. How does protein purification help in the real world? What's the larger picture?

    2. I preformed a GOT Assay, GDH(Glutamate Dehydrogenase) Assay, Folin-Ciocalteau Assay, passed my protein mixture through a Carboxymethylcellulose column,and SDS-Page. How do these things help me purify my target protein (GOT).

    3. What is supposed to occur after each day. (Day One GOT,GDH assays, Day two Folin Assays, Day three Carboxymethylcellulose chromatography, day four SDS-page.)

    4. What do I hope to learn and when is this type of experiment done?

    These questions are really giving me a hard time. Please let me know if anything is unclear.

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    Solution Preview

    Protein Purification Questions
    I did a multiweek experiment doing a protein purification of Glutamate-Oxaloacetate Transaminase (GOT). I have a few questions about my experiment.

    1. How does protein purification help in the real world? What's the larger picture?
    There is a popular adage in Biochemistry which states "Do not waste pure thoughts on an impure protein". It implies that protein purification should precede any biochemical study involving the protein of interest. For example, if you plan to do characterization studies (knowing the optimum pH, optimum temperature, molecular weight and isoelectric point of the protein), kinetic studies (determining the Km and Vmax) or sequencing studies (knowing the primary structure of the protein), you must make sure that you are working on a very pure protein. Presence of contaminating proteins will interfere with the behavior/properties of the protein of interest, making your results inaccurate.
    In addition, obtaining a highly pure protein is a must in industries. Manufacture of vaccines requires the isolation of only the antibody (a protein) that is specific to a particular antigen. In medicine, isolation of insulin (a protein) in highly pure form is important in the treatment of diabetes. In food industries, isolation of novel amylases will allow the efficient conversion of starch to sugar. These are just a few of the important applications of protein purification. It is not simply an academic exercise. It has practical importance.

    2. I preformed a GOT Assay, GDH(Glutamate Dehydrogenase) Assay, Folin-Ciocalteau Assay, passed my protein mixture through a Carboxymethylcellulose column,and SDS-Page. How do these things help me purify my target protein (GOT). ...

    Solution Summary

    Protein purification in the real world are examined. How to target specific proteins are determined.

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