Protein IV Flashcards
dialysis
separates based on size
dialysis elution
large molecules retained, small molecules diffuse out
centrifugation
separates based on size and density
centrifugation elution
larger, more dense proteins sediment faster
size exclusion (gel filtration) chromatography
separates based on size
size exclusion chromatography elution
large proteins elute first, small proteins elute last; small molecules get stuck in porous beads
anion exchange chromatography
separates based on negative charge
anion exchange elution
positively charged proteins will not bind and elute first; most negatively charged elute last
pH < pKa
positive charge
cation exchange chromatography
separates based on positive charge
cation exchange elution
negatively charged proteins do not bind and elute first; the most positive will elute last
affinity chromatography
separates based on specific interaction with protein
affinity elution
only specific protein will bind to column
HPLC
higher resolution, better separation
gel electrophoresis
separates based on size
gel electrophoresis elution
small DNA molecules run faster than large molecules
SDS-PAGE
denatures protein for uniform charge; separates based on size
SDS-PAGE elution
small proteins faster than large
isoelectric focusing
separates based on pKa
isoelectric focusing elution
proteins with highest pKa run closest to negative electrode; proteins with lowest pKa run closest to positive electrode
western blot
detects specific proteins
western blot info
proteins are separated by SDS-PAGE prior to blot
ELISA
detects specific proteins
ELISA info
proteins are not separated
peptide fingerprinting
detects peptides from proteolytic digestion of protein
peptide fingerprinting info
separates based on size in one direction and charge in the other
edman degradation
detects amino acid sequence of protein
mass spectrometry
detects molecular mass of protein; can compare to known proteins in database to find what the protein is
NMR spectrometry
detects protein structure and dynamics
x-ray crystallography
detects protein structure
cryo-electron microscopy
detects structure of protein complexes
dialysis in medicine
in renal failure, urea and other small solutes accumulate in the blood –> dialysis is used to remove the small solutes (they can cross dialysis membrane, but blood cells will stay put)
the greater the sedimentation coefficient _
the faster particles will sediment
centrifugation in blood
RBCs will sediment faster, resulting in red blood cells at bottom and plasma at top
predicting elution in size exclusion
weight is determined by total MW (not just subunit) –> a 56.6 tetramer is actually 226.4
anion exchange setup
column is loaded with positively charged anions to trap negative proteins; to displace these bound negative proteins and allow them to elute, Cl- is added
cation exchange setup
column beads are negatively charged to trap positive proteins; Na+ is added to displace the bound positive proteins and allow them to elute
pH < pI
net positive charge
pH > pI
net negative charge
isoelectric focusing in medicine
sickle cell hemoglobin has higher isoelectric point than regular
western blotting setup
SDS-PAGE separates proteins –> transfer to nitrocellulose –> sheet incubated with an antibody that recognizes proteins of interest
ELISA
similar to Western blotting but proteins are bound to a solid support
direct ELISA
reporter enzyme is coupled directly to the primary antibody
indirect ELISA
reporter enzyme is coupled to secondary antibody
capture assay ELISA
an antibody is bound to the solid surface then the antigen is added –> to detect antigen a primary antibody is added –> excess antibody is washed off –> secondary antibody is added –> washed off –> color reaction
polyclonal antibodies
mixture of several antibodies that are usually produced by different B cell clones of an animal
monoclonal antibodies
antibodies generated from identical B cells that recognize a single epitope
polyclonal antibody formation
inject antigen into an animal –> after immunization, polyclonal antibodies can be obtained directly from serum
monoclonal antibody formation
B-lymphocytes are isolated from the animal’s spleen and fused with a myeloma cell line –> immortal B-cell-myeloma-hybridomas –> grow continuously in culture producing antibodies
in isoelectric focusing, as pH increases _
proteins with low pIs will stop moving; those with highest pI’s will stop moving at highest pH
