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| fluoresence from which fluorophore is actually being measured |
NBD (green)
higher when unquenched and not as close together |
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mcs5109 Wed, 27 Jan 2010 08:27:50 GMT |
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| Which fluorophore does the quenching and which is quenched? |
detects for NBD forescence (green)
quenched if too tight together |
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mcs5109 Wed, 27 Jan 2010 08:27:50 GMT |
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| what are the two fluorophores used to do FRET in these experiments |
NBD and rhodamine |
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mcs5109 Wed, 27 Jan 2010 08:27:50 GMT |
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| SNARE energy |
The SNAREs form a 4 stranded coiled-coil and a very tight “zipping up” of two membranes
where the energy for fusion comes from. |
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mcs5109 Wed, 27 Jan 2010 08:19:04 GMT |
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Name the t-SNAREs that act in synaptic
vesicle fusion |
syntaxin and Snap25 |
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mcs5109 Wed, 27 Jan 2010 08:19:04 GMT |
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| Name the v-SNARE that acts in synaptic vesicle fusion. |
synaptobrevin |
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mcs5109 Wed, 27 Jan 2010 08:19:04 GMT |
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| If a protein wants to know whether it is in the nucleus what does it need to recognize? |
?? |
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mcs5109 Wed, 27 Jan 2010 08:19:04 GMT |
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Is [Ran-GTP] higher in the nucleus or the cytoplasm?
Is [Ran-GDP] higher in the nucleus or the cytoplasm? |
•RanGTP is present in the nucleus (due to action
of GEF called RCC1) |
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mcs5109 Wed, 27 Jan 2010 08:00:07 GMT |
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| What is the GAP specific for Ran? |
Rna1p and RanBP1
causes Ran to hydrolyze GTP |
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mcs5109 Wed, 27 Jan 2010 08:00:07 GMT |
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What is the GEF specific for Ran? () where is it? What does it bind? Why is
this important? |
Rcc1
-in the nucleus
- |
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mcs5109 Wed, 27 Jan 2010 08:00:07 GMT |
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which component of the dimeric receptor binds the nucleoporin protein in the nuclear
pore? |
•Importin-α/β/ cargo complex binds nucleoporin
via importin-β |
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mcs5109 Wed, 27 Jan 2010 08:00:07 GMT |
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| which component of the dimeric (alpha/beta) receptor binds the NLS? |
•Importin-α/cargo binds importin-β |
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mcs5109 Wed, 27 Jan 2010 08:00:07 GMT |
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| what does a classical NLS look like? |
•4 to 8 amino acid
-rich in positively charged amino acids
•NLS can be situated anywhere in
the protein
mutation of a NLS can demonstrate that the NLS is necessary for nuclear localization
transfer of a NLS to a gold bead is an experiment that can demonstrate that a NLS is
sufficient for nuclear localization. Understand how this is not the same thing |
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mcs5109 Wed, 27 Jan 2010 07:48:38 GMT |
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| importin alpha and beta |
form the heterodimeric nuclear import receptor for proteins with
a classic NLS. |
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mcs5109 Wed, 27 Jan 2010 07:44:36 GMT |
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| What proteins are capable of crossing the nuclear membranes? |
-must have nuclear localization signal
-or a small protein, less than 60kD |
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mcs5109 Wed, 27 Jan 2010 07:44:36 GMT |
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| nulear PM info |
-double membrane
-perforated by nuclear pores
-lumen is continuous with the ER lumen
-phospholipid bilayer curves around at the nuclear pore |
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mcs5109 Wed, 27 Jan 2010 07:44:36 GMT |
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| NaF |
inhibits glycolysis. |
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mcs5109 Wed, 27 Jan 2010 07:23:23 GMT |
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| Dinitrophenol |
uncoupler - dissipates proton gradient and reduces ATP by >80%. |
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mcs5109 Wed, 27 Jan 2010 07:23:23 GMT |
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| TCA precipitation assay protocol |
-incubate cells in 3H-leucine, which will be incorporated into proteins.
-precipitate proteins using TCA.
• TCA precipitates proteins and nucleic acids but not amino acids and
nucleotides.
• Measure %radioactivity that is incorporated into proteins (precip/precip+sup)
• Compare this ratio in presence and absence of inhibitor
%TCA -%TCA w/ inhibitor / %TCA |
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mcs5109 Wed, 27 Jan 2010 07:19:20 GMT |
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| Assaying potency of the inhibitors - |
TCA precipitation |
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mcs5109 Wed, 27 Jan 2010 07:19:20 GMT |
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| What do cyclohexamide and puromycin do? What idea is being tested by using these? |
puromycin or
cyclohexamide (protein synthesis inhibitors). |
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mcs5109 Wed, 27 Jan 2010 07:19:20 GMT |
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| Why do human antigens appear to be moving faster? |
cause |
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mcs5109 Wed, 27 Jan 2010 07:19:20 GMT |
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| mouse/human colors |
Mouse: flourescen (green)
Human: rhodamein (red) |
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mcs5109 Wed, 27 Jan 2010 07:12:32 GMT |
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| What proteins are recognized by the antibodies in the alloantiserum? |
HUman: VA-2 cells
Mouse: MHC molecules |
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mcs5109 Wed, 27 Jan 2010 07:09:31 GMT |
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| How were nonspecific antibodies removed from each population? |
pre-clear |
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mcs5109 Wed, 27 Jan 2010 07:09:31 GMT |
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| What is a cell line? |
To maintain cells in culture requires that the cells be immortalized so that they can
proliferate almost indefinitely rather than dying out. We often call these
immortalized cells a “cell line”. |
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mcs5109 Wed, 27 Jan 2010 07:01:01 GMT |
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Where are sugar
modifications found on transmembrane proteins (outside of the plasma membrane or
inside, lumenal side of Golgi or cytoplasmic side)? |
Glycosylated on the noncytoplasmic face |
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mcs5109 Wed, 27 Jan 2010 06:54:18 GMT |
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Where are Golgi membrane proteins integrated into the bilayer? What about
mitochondrial proteins? |
Mitochondria and peroxisomes - posttranslational transport.
• ER - cotranslational transport (MBOC 12-44 and 12-45). |
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mcs5109 Wed, 27 Jan 2010 06:54:18 GMT |
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Does the ER lumenal or cytoplasmic portion of a protein
destined for the plasma membrane end up on the cell surface? |
N-terminus in the ER lumen will end up on the cell surface
• N-terminus in the cytoplasm will end up in the cytoplasm |
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mcs5109 Wed, 27 Jan 2010 06:54:18 GMT |
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What organelles can insert proteins with an alpha-helical transmembrane protein into the
bilayer? |
golgi, ER? |
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mcs5109 Wed, 27 Jan 2010 06:54:18 GMT |
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| What is an integral membrane protein? (How could you dissociate an integral membrane protein from a lipid bilayer in an experiment?) |
require detergent to dissociate
• Integral membrane proteins are attached through some kind of hydrophobic connection
with hydrophobic core of the bilayer. |
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mcs5109 Wed, 27 Jan 2010 06:29:49 GMT |
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| What is a peripheral membrane protein? (How could you dissociate a peripheral membrane protein from a lipid bilayer in an experiment? |
dissociate with high salt or pH changes.
• Associations involve ionic interactions but not covalent interactions. |
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mcs5109 Wed, 27 Jan 2010 06:29:49 GMT |
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| Micelle? |
sequestering the hydrophobic single tail regions in the micelle centre. |
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mcs5109 Wed, 27 Jan 2010 06:26:30 GMT |
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| What effect will SDS have on a lipid bilayer? ... on a transmembrane protein? |
SDS denatures and separates subunits - inactivates
Detergents are themselves amphipathic molecules, and this allows them to coat
hydrophobic surfaces |
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mcs5109 Wed, 27 Jan 2010 06:26:30 GMT |
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Where is most of the cellular cholesterol found?
What effect does it have on the membrane? |
-PM
-thicker and less permeable |
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mcs5109 Wed, 27 Jan 2010 06:19:41 GMT |
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| why can cholesterol "flip-flop" while phospholipids can't? |
Because of the limited hydrophilicity of the head group,
cholesterol is the one lipid that can flip from one leaflet to
another without the requirement of a special transporter |
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mcs5109 Wed, 27 Jan 2010 06:19:41 GMT |
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| Less abundant components are |
cholesterol -
inositol phospholipids |
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mcs5109 Wed, 27 Jan 2010 06:19:41 GMT |
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| Major membrane lipid components are |
phosphatidylserine
phosphatidylcholine
phosphatidylethanolamine
sphingomyelin |
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mcs5109 Wed, 27 Jan 2010 06:18:05 GMT |
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| What is a leaflet? |
Each layer of a membrane bilayer is often called a leaflet |
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mcs5109 Wed, 27 Jan 2010 06:18:05 GMT |
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