Non-genetic analysis of gene function

Where is a protein in the cell? In the organism?
1. Antibodies
  1. To make a protein specific antibody we first need to make a lot of the protein:
    1. Expression vectors use a bacteriophage promoter to drive high levels of RNA synthesis.
      1. The promoters have to be inducible, otherwise the bacteria would die rapidly.
      2. Chemicals or temperature shifts are used to induce protein expression. After induction, the bacteria are harvested and lysed to make a crude extract.
        Expression plasmids often include a epitope tagging system that allows for rapid and efficient purification of the protein. The tag is fused in-frame to the cDNA during the cloning.
  2. Antibody-affinity purification
    1. Epitope tags are peptides for which antibodies are already available.
      1. Bacterial cells are lysed and the resulting solution (crude extract) is poured onto a column
        Small beads about the size of sand grains have been coated with the antibody
  3. Making a protein specific antibody:
    1. Antibodies bind to small regions of proteins with very high specificity - called an epitope
      1. Tagged antibodies have dyes or enzymes attached (conjugated) to them so that we can determine their location
  4. Detection Methods
    1. Some conjugates are dyes that are fluorescent allowing us to detect the location using specific wavelenghs of light
      1. Sometimes we use antibodies to examine the sub-cellular localization of the protein, other times we are testing where is the protein in the organism (which tissues)
        Commonly used enzyme conjugates:
        alkaline phosphatase - substrate turns blue
        horseradish peroxidase - substrate turns brown
        Enzyme detection can enhance sensitivity
        Often we make an two antibody sandwich, this amplifies the signal. This is because many 2º antibodies bind to each 1º antibody:
  5. How to use antibodies:
Where is a gene transcribed in a tissue or organism?
1. RNA in situ
Visualizing gene expression and protein localization in living cells
1. GFP
2. Uses for GFP transgenic lines:
  1. To follow expression of a gene in the whole animal.
    1. To answer: What tissue is the gene expressed in?
  2. To follow subcellular localisation of a protein
    1. For example some transcription factors become nuclear upon activation.
      1. This happens after the cell receives a signal
        Use a GFP fusion for this
        GFP fusion usually does not alter the activity of the protein.
  3. To follow the behavior of cells in vivo.
    1. The GFP line is used to mark specific cells so that they can be distinguished from their neighbours.
      1. Use a GFP reporter construct for this.

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Non-genetic analysis of gene function

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	Criado por Kristi Brogden mais de 10 anos atrás