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exam 2 content
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what were the components of the electrophoresis buffer?
25 mM
192 mM
0.1% SDS
what were the components of the transfer buffer?
25 mM tris
192 mM glycine
20% methanol
what does SDS-PAGE stand for?
sodium dodecyl sulfate polyarcylamide gel electrophoresis
how does SDS-PAGE work?
SDS binds and confers negative charge to proteins
SDS uniformly binds proteins
what happens when an electrical field is applied to a PAG?
the neg. charged proteins are moved through the polyacrylamide matrix toward the anode
migration rate depends on size of proteins
what are the membrane types used in western blotting applications?
polyvinylidene fluoride (PVDF) and nitrocellulose
can be done using wet or semi-dry transfer
is wet or semi-dry faster?
semi-dry
requires less volume of reagents
produces comparable results with the wet transfer system
wet transfer less prone to failure due to drying of membrane for large proteins
what stain is used?
ponceau-S red or copper phthalocyanine tetrasulfonic acid
timings
wet membrane in ethanol for 15 seconds
decant methanol and soak membrane in dH2O for 2 minutes
equilibrate for 5 minutes
transfer at 15 V for 30 minutes, current limit of 5.5 mA/cm2
remove membrane and soak in 100% methanol for 10 seconds
immerse blot in 20% methanol for 5 minutes
set up
cathode (negative)
buffer soaked filter paper (2)
nitrocellulose membrane
buffer soaked filter paper (2)
anode (positive)
other techniques that use antibodies to erect proteins
immunofluorescence → seeing proteins in cells
enzyme-linked immunosorbent assay (ELISA) → IDing biological fluids
how is ponceau staining done?
prior to immunostaining
submerge membrane with stain and put on rocker for 15 mins
remove membrane, rinse with dH2O
capture image
ensures that proteins have transferred from gel to membrane and blotting was successful