electropheresis Flashcards
what is electropheresis
separation of charged molecules under an electric field eg amino acids, proteins
what are the components of electropheresis
power source - direct current - voltage
electropheresis unit - electrode and buffer reservoir and insulating cover - prevents evaporation
buffer - no interaction with the sample
what affects electropheresis rate
size up, rate down
charge up, rate up
shape
describe the supporting media of electropheresis
paper - speedy
gel - molecular sieving
polyacrylamide gel - requires initator, pore size manipulated
agarose gel - higher concentration, smaller pore size
what is sds
polyacrylamide gel electropheresis
separation depends on net charge, size and shape of protein
gel concentration affects mobility
what is isoelectric focusing
a ph gradient is created from the buffer mixture - polyampholytes
the electric field migrates proteins to the corresponding isoelectric points on the gradient
proteins form bands and stop moving as isoelectric point is reached
what is 2d gel electropheresis
protein separated by isoelectric focusing
proteins undergo sds, repeated again at 90 degrees - separation of protein by size
the gel is stained - each dot is a protein
what is proteome profiling
two 2d gels compared - differences in spot pattern and intensity - difference in protein amount in the 2 proteomes
how fast does dna move through a pore matrix
if supercoiled dna - moves fast
if relaxed dna - moves slow
what is the snow drop blotting technique
SOUTHERN - DNA - agarose gel, horizontal, nucleic acid complementary to target dna
NORTHERN - RNA - agarose gel + formaldehyde, horizontal, nucleic acid complementary to target dna
O - O
WESTERN - PROTEIN - acrylamide gel, vertical, antibody binds to protein epitope
all are fluorescent and radioactive
