lec 1 - protein purification Flashcards
Aromatic amino acids absorb at
218 nm
special group of proteins called ______ that bind proteins
lectins
metallic proteins cause problems for
purification schemes, EDTA inactivates metal ions
when pH = pI
the protein is uncharged and repels itself
frothing
denaturing a protein by causing it to be close to an air bubble (the hydrophobic core binds to the air), the protein denatures then precipitates
purification considerations
- trade between yield and purity
- active or inactive
- expense (complicated purification schemes more expensive)
- time (proteins denature)
- need an assay (purification needs fractionation)
- source material (liver has little myoglobin)
- extract must be clear
- protein must be soluble
salting out
at high salt proteins precipitate
at low salt
proteins repel each other
when adding salt
charges are neutralized and proteins are much more likely to stick together
SDS PAGE
stains with coomassie blue
seperates on basis of size
first sds denatures protein
alkaline chain
binds hydrophobic patches on protein
with positive electrode on the bottom gets forced through acrylamide gel
(positively charged residues{histones} behave weirdly on polyacrylamide gel because they bind more protein and move slower
using tags in affinity purification
add a tag that binds your favourite protein, a ligand that binds the tag, and a bead that binds the ligand, a protease can then cut the protein-tag site(add protease, wash, incubate and then wash your protein off, can have problems if the protein your trying to purify is a protease
how structures are generally determined
getting the gene your interested in, (can put it in a vector under the control of an inducible promoter or use a tag)
Some tags
Histidine(6)(has high affinity for nickel or cobalt)
flag(so you can use an anti-flag antibody)
a whole protein(like glutathion-s-transferase(if your protein is unstable or insoluble make it an FST fusion protein and then its more stable))
considerations with proteases
if there is a site in the protein with the same amino acid sequence as the cleavage site
