HW 2 BICD 110

HW 2 BICD 110 - 1 What is fluorescence Describe two methods...

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1. What is fluorescence? Describe two methods how fluorescence microscopy can be used to localized proteins within cells. What are the advantages and disadvantages of the respective methods? a. A chemical is said to be fluorescent if it absorbs light at one wavelength (the excitation wavelength) and emits light (fluoresces) at a specific and longer wavelength of different color b. Immunofluorescent microscopy b.i. Can detect specific proteins in fixed cells b.i.1. Grow cells on cover slips b.i.2. Fix cells with paraformaldehyde b.i.3. Permealize cells with detergents b.i.4. Incubate cells with primary antibody specific for protein of interest b.i.5. Incubate cells with secondary antibody specific for primary antibody b.i.6. Observe cells under the fluorescence microscope b.i.7. Fluorescence is often brighter b.i.8. Specifically localize antibodies to endogenous proteins b.i.9. Disadvantage: kill cell/fix b.i.9.a. Problems with fixation formaldehyde/glutaraldehyde is different b.ii. antibody is covalently linked to a fluorochrome
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HW 2 BICD 110 - 1 What is fluorescence Describe two methods...

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