bis_104_answers_to_ldl_fh_qs_fall_l_08

bis_104_answers_to_ldl_fh_qs_fall_l_08 - Possible cellular...

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Possible cellular defects in the binding and processing of LDLs. 1. LDL-R is not synthesized at all. Strategy #1: Direct assay for LDL-R synthesis. 1. Add aa isotopes to culture of cells from FH- individuals; allow time for uptake and incorporation of aa into protein. 2. make a total protein extract of cells and prep for SDS-PAGE. 3. run SDS gel and then transblot (western). 4. probe blot: autoradiography to reveal all labeled proteins Probe with labeled abs vs LDL-R or with labeled LDLs to locate LDL-R 5. examine for co-incidence of radio-labeled protein / LDL-R (should run in parallel experiment using FH+ cells) Strategy #2: Indirect 1. culture FH- cells 2. add fluor-labeled anti-LDL-R ab to culture; allow time for binding 3. wash away unbound ab and observe cells for PM fluorescence by fluor microscopy Could also permeabilize cells and apply ab as above to determine if there is cytoplasmic LDL-R. 2.
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bis_104_answers_to_ldl_fh_qs_fall_l_08 - Possible cellular...

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