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BIS 104 PQ 18 ans spr 2010

BIS 104 PQ 18 ans spr 2010 - BIS 104 PQ 18 ans spr 2010 18...

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BIS 104 PQ 18 ans spr 2010 18. Using the technique of IHC and fluorescence microscopy, Mukouyama has shown that arterial ECs begin to express ephrinB2 on their plasma membranes sometime between E13 and E15 in mouse limb skin. a. Use SDS-PAGE and western blotting to test the hypothesis that ephrinB2 is present in the plasma membrane of arterial ECs on day 15, but not on day 13. 1. Obtain limb skin tissue samples from E13 and E15 mice. 2. Dissociate tissue into single cells. 3. Obtain enriched population of ECs from the cell mix by using FACS to purify ECs (use PECAM-1 as cell- specific target for fluorescent marker). Positive control: examine a sample of EC fraction by phase-contrast and by fluorescence microscopy to assess degree of purity. (Do they look like ECs? Are they PECAM-1 positive?) 4. Lyse and fractionate ECs to obtain an enriched PM preparation. Positive control: sample and examine by TEM to confirm purity of PM prep. Or test for PM-specific enzymes and absence of contaminants. (that is, is the prep free of nuclei, mitochondiria, ER, etc?)
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