MCB 253 Final Exam
blocking buffers - ✔✔Used to prevent nonspecific binding interactions between the antibodies and
nitrocellulose membrane. Added after transfer to nitrocellulose before antibody staining.
10X TBST: designed for washing the blots in between stains and 5%nonfat dried milk.
bromophenol blue - ✔✔ph marker. used to indicated when the protein reached the bottom of the
electrophoresis
Coomassie Blue - ✔✔a dye that stains proteins blue and allows them to be visualized. it binds to arg, lys,
his residues and is then stable in its anoinc form
only used in sds page procedure not used for the sds part of the western blot
DAPI - ✔✔nuclear localizing dye that binds to the AT rich sequences of dsDNA in the minor groove
- blue @ 358nm
fluorophore intercalating agent
explain the difference in visualization methods of SDS PAGE and western blot - ✔✔SDS PAGE uses
coomassie blue which prevents further use of the protein.
western blot - HRP color development which binds to the secondary AB following AB treatment.
fluorophore - ✔✔fluorescent chemical compound that can re-emit light upon light excitation. a laser
excites the fluorophore causing electrons to be releases and light to be emitted
formeldehyde - ✔✔Kills cells, but keeps everything intact with covalent bonds so cells do not move.
, Gel is on cathode (-) and
nitrocellulose membrane is on anode (+). - ✔✔--
glycerol - ✔✔in SDS, it is added to the protein sample and acts to weigh down the sample. restricts
overflow and uneven gel loading
glycine in the cellular localization experiment - ✔✔reacts with formaldehyde and reduces background
noise as well as bind to the stain molecules that don't attach to the membrane and makes the image
clearer
how can you confirm that your protein sample is in fact the specific protein with a native gel? - ✔✔the
charge and isoelectric point will be the same
how can you confirm that your protein sample is in fact the specific protein with a western blot? -
✔✔the same antibody will bind to the epitope
how can you confirm that your protein sample is in fact the specific protein with SDS PAGE? -
✔✔molecular weight is same
How does fluorescence microscopy work? - ✔✔specimen (dye) absorbs light, to an excited state. since
this energy can't be contained for too long, there is an emission of light energy, thus the fluorescence
we see.
How does SDS buffer work? - ✔✔-SDS buffer denatures the hydrophobic interactions of proteins and
gives them an overall negative charge bc SDS is an anionic detergent
-thus proteins have the same charge to mass ratio, meaning that the bigger the protein, the more
negative it is.
-therefore proteins migrate in the gel only on the basis of mass
How does SDS-PAGE work? - ✔✔after the anionic detergent (sds buffer) attached to the proteins to give
them a net negative charge, the gel is run and the proteins will move vertically from the (-) cathode to
blocking buffers - ✔✔Used to prevent nonspecific binding interactions between the antibodies and
nitrocellulose membrane. Added after transfer to nitrocellulose before antibody staining.
10X TBST: designed for washing the blots in between stains and 5%nonfat dried milk.
bromophenol blue - ✔✔ph marker. used to indicated when the protein reached the bottom of the
electrophoresis
Coomassie Blue - ✔✔a dye that stains proteins blue and allows them to be visualized. it binds to arg, lys,
his residues and is then stable in its anoinc form
only used in sds page procedure not used for the sds part of the western blot
DAPI - ✔✔nuclear localizing dye that binds to the AT rich sequences of dsDNA in the minor groove
- blue @ 358nm
fluorophore intercalating agent
explain the difference in visualization methods of SDS PAGE and western blot - ✔✔SDS PAGE uses
coomassie blue which prevents further use of the protein.
western blot - HRP color development which binds to the secondary AB following AB treatment.
fluorophore - ✔✔fluorescent chemical compound that can re-emit light upon light excitation. a laser
excites the fluorophore causing electrons to be releases and light to be emitted
formeldehyde - ✔✔Kills cells, but keeps everything intact with covalent bonds so cells do not move.
, Gel is on cathode (-) and
nitrocellulose membrane is on anode (+). - ✔✔--
glycerol - ✔✔in SDS, it is added to the protein sample and acts to weigh down the sample. restricts
overflow and uneven gel loading
glycine in the cellular localization experiment - ✔✔reacts with formaldehyde and reduces background
noise as well as bind to the stain molecules that don't attach to the membrane and makes the image
clearer
how can you confirm that your protein sample is in fact the specific protein with a native gel? - ✔✔the
charge and isoelectric point will be the same
how can you confirm that your protein sample is in fact the specific protein with a western blot? -
✔✔the same antibody will bind to the epitope
how can you confirm that your protein sample is in fact the specific protein with SDS PAGE? -
✔✔molecular weight is same
How does fluorescence microscopy work? - ✔✔specimen (dye) absorbs light, to an excited state. since
this energy can't be contained for too long, there is an emission of light energy, thus the fluorescence
we see.
How does SDS buffer work? - ✔✔-SDS buffer denatures the hydrophobic interactions of proteins and
gives them an overall negative charge bc SDS is an anionic detergent
-thus proteins have the same charge to mass ratio, meaning that the bigger the protein, the more
negative it is.
-therefore proteins migrate in the gel only on the basis of mass
How does SDS-PAGE work? - ✔✔after the anionic detergent (sds buffer) attached to the proteins to give
them a net negative charge, the gel is run and the proteins will move vertically from the (-) cathode to
