L13 Flashcards
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
whats the acrylamide stacking gel concentrations
top 5%
bottom 12%
whats the top layer of a stacking gel
the rolling start gel
whats the bottom gel in stacking gel
race zone gel
what is the gel in stacking gel
TRIS HCL
what is the buffer used in stacking gel
glycine
