BIOL 4285 Lecture Notes - Ecori, Doxycycline, Homologous Recombination

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amethysthyena552

16 Oct 2011

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In the 70s/80, the models people used for cancer were birds. Translocation of chromatin can lead to activation of oncogenes. No one knew the molecular events that caused cancer in birds via viruses. Experiment: took a virus that causes tumours in chickens and took the tumour cells from a chicken and introduced them into a rabbit (implanted under the skin) Rabbit would break down the cells and make antibodies against the proteins present in tumour cells. When they put normal avian cells into rabbits, they would get an array of proteins that were indicative of the antibodies raised against those cells. When the put in the tumours, they got extra bands/proteins that they then purified. Got three main bands and called them the small t antigen, middle t antigen and large t antigen. Took antibodies that the rabbit produced against these three proteins the rabbit and probed the proteins in the avian species.

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Seperation and detection of proteins 1. What was the purpose ofwashing the red blood cells with saline prior to lysis? 2. Whatproperty of the lysis buffer caused the red blood cells to lyse? 3.What are the differences between the primary and secondaryantibodies used in this study? 4. What are the advantages of usingindirect immunodetection over direct immunodetection? 5. Antibodiesare made when animals detect a foreign antigen. All mammals carryalbumin in their blood. How was a rabbit antibody against albumincreated? Was this lethal to the rabbit? Explain you reasoning. 6.Albumin is not a cytoskeletal protein. What was the purpose ofusing antibodies to detect albumin in our cytoskeletalpreparations? 7. Why was it necessary to transfer the proteinsseparated by SDS-PAGE onto nitrocellulose membranes prior toincubation with anti-albumin antibodies? 8. Why did weelectrophoretically transfer the proteins resolved by SDS-PAGE ontonitrocellulose? Can you suggest another way to transfer proteinsfrom a SDS gel to a membrane? 9. Why did we wash the nitrocelluloseblots with blotting buffer three times after incubating with theprimary and secondary antibodies?

scarletalpaca360

When producing insulin, the gene for its production is insertedinto the plasmid of a bacterium that is allowed to replicatefreely.

Why can't the same thing be done with antibodies (as Iunderstand it, currently cancer cells are fused with mammaliancells that produce these antibodies, and now the tumour is allowedto replicate, much like the bacterium, making more antibodies)?

What is different about these two proteins that means fairlydistinct processes of production are required for each?

Antibodies raised against proteins of interest are useful toolsfor biomedical research, because they allow the visualization orisolation of individual proteins from a complex mixture ofdifferent proteins. A Western blot (WB) is one method ofvisualizing proteins: all proteins are extracted from cells, theproteins are denatured and separated by size using electrophoresisthrough a gel, and the protein of interest is detected by bindingto an antibody that is linked to a fluorescent or luminescentmolecule. Immunofluorescence (IF) provides a way to visualize aparticular protein within a cell using microscopy: the cell is a"fixed" (killed and preserved intact), flooded with an antibodythat binds a protein of interest, and washed free of unboundantibody. Because the antibody is also bound to a fluorescentmolecule, the subcellular location and intensity of thefluorescence indicates the location and concentration of theprotein of interest. Unfortunately, many antibodies that work wellfor WB are not useful for IF. Consider an antibody that yields anintense signal located at the expected position on a WB. When theWB is performed with cells lacking the protein of interest, theintense signal is absent but there remain a few faint signals fromnon-specific proteins of the wrong size. Yet this antibody is notuseful for IF because in IF the wild-type cells and the cellslacking the protein of interest look the same. Suggest a possibleexplanation for the observation that the antibody is more specificin a WB than in an IF experiment; note that at least fiveexplanations are possible.

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BIOL 4285 Lecture Notes - Ecori, Doxycycline, Homologous Recombination

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