biochem 003 techniques in protein biochemistry part 2 (proteome protein purification chromatograph

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  1. after incubating the protein mixture from the rat uterus with radioactive estradiol and centrifuging the mixture, what must one do?
    after the centrifugation is complete, a small hole is made in the bottom of the centrifuge tube and portions of the gradient are collected and tested for radioactivity. The estradiol-estrogen receptor complex should be situated at about 4S. 

    However, there are other proteins in the mix that have an S value of about 4S, therefore, the fraction containing the complex also contains other proteins that need to be discarded. We can separate the complex from the other protiens by using immunological techniques
  2. what are polyclonal antibodies?
    antibodies that are specific for the same antigen, but different epitope
  3. what are monoclonal antibodies?
    antibodies that are specific for the same antigen and the same epitope
  4. how can one create an immortal cell line that produces monoclonal antibodies?
    immortal cell lines producing monoclonal antibodies can be generated by fusing normal antibody producing cells with cells from a type of cancer called multople myeloma
  5. Describe the immunological technique that can be used on the mixture of proteins in order to obtain estrogen receptor proteins
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  6. describe the indirect ELISA test
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  7. describe the sandwich ELISA test
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  8. how is western blotting done?
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  9. how is the amino acid composition of a protein determined?
    the protien is hydrolyzed, and the constituent amino acids are separated on an ion exchange column. The amino acids are visualized by reaction with fluorescamine
  10. how can the amino acid sequence of protein be found?
    by using Edman degradation
  11. Describe how Edman degradation is done
    the protein is exposed to PTH, which reacts with the N-terminal amino acid to form a PTH-derivative. The PTH-amino acid can be released without hydrolyzing the the remainder of the protein, and the degradation is subsequently repeated.
  12. what is a disadvantage of the Edman degradation? how can one work around this?
    Because the reactions of the Edman degradtion procedure are not 100% effective, it is not possible to sequence polypeptides longer than 50 amino acids

    In order to sequence the entire protein, the protein is chemically or enzymatically cleaved to yield peptides of fewer than 50 amino acids.

    The peptides are then ordered by performing a different cleavage procedure in order to generate overlap peptides
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biochem 003 techniques in protein biochemistry part 2 (proteome protein purification chromatograph
2014-10-06 00:11:44
biochem 003 techniques protein biochemistry part proteome purification chromatography SDS PAGE isoelectric focusing ELISA
biochem 003 techniques in protein biochemistry part 2 (proteome, protein purification, chromatography, SDS PAGE, isoelectric focusing, ELISA) #6
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