Anode, cathode - Anode (+) membrane <--- cathode (-) gel.
B-metacaptoethonol - breaks disulfide linkages
Blocking buffer. Types? - Used to prevent nonspecific binding interactions between the antibodies
and nitrocellulose membrane. Added after transfer to nitrocellulose before antibody staini...
Blocking buffer. Types? - ✔✔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
Continous gel - ✔✔gel and tank buffers are the same, single phase gel. Examples are PAGE, agaores and
starch gels
DAPI binds what? - ✔✔Most stable. Nucleus, is a flourophore. Binds minor groove of DNA AT clusters.
Describe a stacking and resolving gel. An SDS discontinous gel. - ✔✔The top is the stacking gel which
includes the sample wells. It is responsible for compressing (stacking) the proteins into thin layers when
they reach the revolving gel. Usually much lower percent (4%)
The revolving gel is the bottom which resolves them based on their size to charge ratio (12%).
Describe electroblotting - ✔✔movement of proteins from western to nitrocellulose.
Describe how the microscope works - ✔✔So shine a light and a filter (dichroic mirror) will absorb all
wavelengths that are not the exciting wavelength will allow passage of only a certain wavelength of the
light through to tough the sample. The e- in the sample are excited and flouresce and the light given off
goes through another filter that only absorbs
, Discontinous - ✔✔gel and tank buffers are different. Two phase gel, stacking gel, example is page.
Discontinous vs continous gel - ✔✔Discontinous (gradient): 4-20%. good for if the protein is an unknown
weight
Continous: 10 or 12%: allows for better resolution of particular protein weight ranges.
Flouorescence - ✔✔emmision of a detectable wavelength of light
Fluorophore - ✔✔molecule that can be maximally excited as a particular wevelength and which emits a
fluorescent wavelength at another
Glycien is used in - ✔✔glyciene is used in flourescence microscopy to quench aldehydes that were
unreacted.
Glyciene - ✔✔reacts with excess of formaldehyde from fixation, reduces background noise,
interference.
how big for 12% gel how big for 10% gel - ✔✔10-70 kDa. 15-100 kDa for 10%
How do you image a protein gel? - ✔✔Use coomassie blue dye: it binds to arg, lys, his residues and is
then stable in its anoinc form and is blue.
Immunofluorescence - ✔✔fluorescence by antibodies tagged with a fluorescent molecule
In a native gel, what will convince you that your unknown protein is actually A and not B? - ✔✔Native
gel: knowledge about the natural conformation and intrinsic charge of the two proteins and the results
obtained in relation to knowledge about conformation
In SDS PAGE what will convince you that your unknown protein is actually A and not B? - ✔✔Accurate
MW
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