Chapter 3 exploring proteins and proteoms Flashcards
The assay in protein purification measures
enzyme activity
NADH and NAD? which absorbs light at
340 nm NADH
specific activity
ratio of enzyme activity to the amount of protein in the mixture
what happens to specific activity when the protein is pure
it stays constant
How are homogenats produced
produced by disruptions of cell membranes
Salting out
when protein solubility is decreased when the salt concentration increases
Dialysis and how it’s used
to separate proteins from small molecules by using semipermeable membranes
Gel-filtration chromatography
separation based on size the proteins will emerge first less volume is accessible
separation based on charge
ion exchange chromatography
negatively and positively charged proteins can be separated by
anion exchange (diethylaminoaethyl) cation exchange (carboxymethyl)
His-tags are added in which technique
affinity chromatography
immobilised nickelI and other metal ions can be used
to elute the protein, excess imidazole is used to displace the histidine-tags
polyacylamid gel and why is it a good choice in electrophoresis
formed by the polymerisation of acrylamide with a small amount of cross-linking agent methylenebisacrylamide
B-mercaptoethanol
reduces disulfid bindinger
in electrophoresis the mobility of most polypeptides is proportional to
the logarithm of the masses
what molecules are used in isoelectric focusing
polyampholytes (small multicharged polymers) having many different pI values
the ….. the value of s the slower a molecule moves in a centifual field
smaller
a ……. dense particle moves more rapidly than a …… particle
more dense
less dense
the buoyant force is smaller for the more dense particle
gradient centrifugation
separation of proteins with different sedimental coefficients
sedimentation equilibrium is useful
because it allows the determination of the mass of for example multimeres
polyclonal why are they useful in protein detection
derived from multiple anti-body producing cell populations. Because protein of low abundance can be bound by multiple antibodies that recognise different antigenic determinants
hybridoma cells how and why
by the fusion of antbody producing cells with myeloma cells
to produce many antibodies with desired specificities
enzyme linked immunosorbent assays are used to
detect and quantify proteins
ELISA what enzymes are used
enzyme linked immunosorbent assays.
horseradish peroxidase
alkaline phosphatase reacts with a colourless substrate to produce a coloured object
types of ELISA
indirect used to detect antibodies
sandwich: used to detect antigens
primary antibodies
antibodies specific for the protein of interest
secondary antibodies
antibodies specific fo the primary antibodies
essential components of mass spectrometers
1- ion source
2- mass analyser
3- detector
mass analyser
the analyte ions are distinguished based on mass to charge ratio
Time of flight mass analyser
ions are accelerated through an elongated chamber under a fixed electrostatic potential
how can the protein ions be broken into smaller peptides
by bombardment with atoms of inert gases such as helium or argon
tandem mass spectrometery
two mass analysers
what molecule is used in Edman degradation
phenyl isothiocyanat
edman degradion is limited to …… residues and why?
50 residues
because not all peptides release the amino acid derivative at each step
disulfied bond can be reduced by and how are they prevented from reforming the bonds
B-mercaptoethanol
dithiothreitol
by being alkylated with iodoacetate that forms stable s-carboxymethyl derivatives
peptide mass fingerprinting
protein cleavage followed by chromatographic separation
what molecules are used in solid phase sythesis of peptides
resin binds to carboxyl-terminal preventing it from reactions with other amino-acids
t-Boc (protecting group of the amino terminal) tert-butyloxycarbonyl can removed by trifluoroacetic acid.
t-Boc
DCC used to activate the aminoacid binding to the carboxyl terminal of the incoming amino acid.
(dicyclohexylcarbodiimid)
the whole peptide is released by HF (hydrofluoric acid)
why are x-rays good in molecular crystollographic studies
because their wavelength corresponds to the lenght of a covalent bond
synchrotron radiation
the acceleration of electrons in circular orbits close to the speed of the light
the spinning of a proton generates
a magnetic momentum
a particular state in NMR is favoured when
the state is alligned with the applied field
what is the reference used in nmr
tetramethylsilane
An interaction between two nuclei is proportional to thr
inverse of the sixth power of the distance between them
