how do you elute affinity chromatography
by semi denaturing proteins using pH
electrophoresis
migration of ions in an electric field
velocity of ions in electrophoresis solution is __________ proportional to charge and ____________ proportional to size and shape
directly
inversely
whats the issue with electrophoresis
when the power is shut off, charged -particles are free to diffuse again
define SDS
a detergent that will denature proteins and give them a negative charge
what contains acrylamide
anything baked, toasted, or fried
what staining is used for visualizing the gel
comaissie blue staining
what 3 dyes can be used for visualizing the gel
comissie blue staining
silver nitrate
fluorescent
if a silver stain is black, what does that mean
that there’s protein
are smaller or bigger proteins more mobile on a gel
smaller
for 1 gel, how to determine size
graphing absolute distance migrated vs log of size of standards
for 2 gel, how to determine size
measure relative distance migrated to compare
relative mobility
how far your protein band goes compared to something fast
what gives a protein size standard curve
plotting Rf vs molecular mass (kD)
as size decreases,________ increases exponentially
mobility
fastest and least sensitive stain for visualizing gel
coomassie blue stain
white on silver staining means what
no protein
why can’t absolute distance be used for 2 gels
because of different conditions (time ran or volts used)
if we are running a gel with large proteins, what concentration of Gel, would be used
low concentration
if we are running a gel with small proteins, what concentration of Gel, would be used
a high concentration gel
tumur supressor protein
p53
p53 runs slower or faster than its true size (44)
slower
4 solutions for overlapping bands
use less volume
concentrate sample
run a vertically longer gel
run a stacking gel
what works really well to separate protein bands
stacking gel
