Biomedical Engineering Reference
In-Depth Information
4
Notes
1. For example, for the studies on polarized EGFR transport in
apico-basolateral traffi cking in epithelial cells, the Madin-
Darby canine kidney (MDCK) cells have been used [
17
].
2. Both retroviral and lentiviral vectors could be used for these
proliferating cell lines.
3. Several other vendors have comparable setups (e.g., Leica
Microsystems, Olympus, Nikon Instruments, and Molecular
Devices).
4. Silencing conditions (amount of reagents used and time of
incubation) need to be optimized. Typically the decrease in
protein level, rather than mRNA, is monitored, as protein
turnover may be much slower, particularly for abundant house-
keeping proteins.
5. If the silencing method targets the coding region, the rescue
construct would need to generate a transcript that will not be
recognized by the si-/shRNA. One could of course make
mutations, but one simple way is to use the cDNA of the
orthologue of another species (e.g., mouse orthologue to res-
cue silencing in human cells). However, if the RNAi method
targets an untranslated region, the coding region itself without
the untranslated parts could be used.
6. Concentration of EGF used may need to be optimized.
Typically, the concentration of EGF used ranges from 0.05 to
0.5
g/ml. It has been shown that the levels of EGF affect
whether clathrin-mediated or clathrin-independent internal-
ization of the ligand-bound receptor predominates, and subse-
quent recycling versus degradation of the ligand-bound
receptor also varies depending on ligand concentration.
7. Biophysical methods, such as those based on surface plasmon
resonance (SPR) and nuclear magnetic resonance (NMR),
require sophisticated equipment and smaller fragments of the
interacting proteins, but could investigate direct physical
associations.
8. This is largely for the purpose of normalization. Not all anti-
bodies can perform immunoprecipitation (and this therefore
needs to be verifi ed), and there is a chance that antibody bind-
ing fortuitously disrupts the interaction being investigated.
9. This is a simple method that generates relatively less volume of
solutions to work with. However, if the acid precipitation
destroys the antibody epitope, other precipitation methods
such as acetone or chloroform/methanol precipitation needs
to be considered.
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