Powerpoint 1 Chapter 3 Flashcards
3D structure of a purified protein can be determined by ? or ?
x-ray crystallography or NMR (and sometimes computationally)
Genome
the blueprint for what proteins may be expressed
Proteosome
the proteins of the genome that are actually expressed
first step in purification
harvest crude protein mixture from whole cell lysates
purification of a protein is based on ? (4)
solubility, size, charge, binding affinity
salting out
purification by increasing salt concentration to draw out proteins as most are less soluble at high salt concentrations
dialysis
purification using a bag with a semipermeable membrane that allows some molecules out (such as salt) while retain some molecules within (such as proteins
Gel-filtration chromatography
proteins passed through column consisting of porous beads made of insoluble but highly hydrated polymers. Large molecules elute first because smaller volume is accessible to them. Small beads diffuse into beads to a greater degree.
Ion-exchange chromatography
Proteins passed through column of negatively or positively charged beads. oppositely charged proteins stick to the beads and elute more slowly. Same charge proteins elute easily.
? beads used for eluting positively charged proteins
carboxymethyl group
? beads used for eluting negatively charged proteins
diethylaminoehtyl
HPLC
enhanced version of column separation techniques that implements a column containing a finer material that requires pressure to be applied for flow to occur. Results in higher resolution and rapid separation.
Gel electrophoresis
a gel with opposite charge on each side will cause proteins to flow in direction of opposite charge. speed of flow based primarily on size and secondarily on charge.
SDS PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sensitive protein separation technique where proteins are separated in denatured form
SDS binding
one SDS binds to every two amino acids. Has negative charge making protein charge contribution insignificant.
SDS PAGE allows separation based entirely upon mass, giving an inverse relationship between ? and ?
mass and mobility
isoelectric focusing
a pH gradient is used to find the isoelectric point of a protein
isoelectric point
pH at which protein charge is zero
two-dimensional electrophoresis
begins with a 1D electrophoresis followed by another 1D electrophoresis 90 degrees to the first
sedimentation coefficient is largely proportional to ?
mass/size
affinity chromatography
purifying proteins based on a highly specific interaction between protein of interest and other chemicals
most common technique for purifying native and recombinant proteins
affinity purification
interactions used in affinity purification
antibody and epitope tag interactions
TAP
tendem affinity purification.
? blue staining
Commassie
A protein purification scheme can be quantitatively evaluated using ?
SDS-PAGE
antigen types (2)
purified protein/partial peptide sequence and epitope tags
antibody structure
two heavy and two lights chains connected by sulfide bonds
polyclonal vs monoclonal antibodies
monoclonal antibodies are all identical and bind to same epitope. Polyclonal antibodies are a heterogenous mixture but also all bind to the same epitope.
hybridoma cells
fusion of short lived antibody producing cell with an immortal myeloma cell
western blot
transfer SDS-PAGE gel to polymer sheet and add radiolabeled specific antibody. Wash to remove unbound radiolabel. Expose and develop to identify protein that interacted with antibody.
two types of ELISA antibody/antigen detection
indirect and sandwich
indirect ELISA
coat well with antigen. wash. allow specific antibody to bind. wash. enzyme linked antibody binds to specific antibody. wash. add substrate that is converted to observable product by enzyme. Color formation is proportional to amount of specific antibody.
sandwich ELISA
coat well with monoclonal antibody. wash. allow antigen to bind. wash. second monoclonal enzyme bound antibody allowed to bind antigen. wash. add substrate that is converted to observable product by enzyme. Color formation is proportional to amount of specific antibody.
common test for HIV uses ?
indirect ELISA
sandwich ELISA commonly used for detection of ?
small amounts of antigen such as hormone
Peptide bonds are hydrolyzed in ?M HCl at ? degrees C
6 M, 100 degrees
amino acid mixture is separated by ? chromatography
ion-exchange
free amino groups react readily with ? or ? to produce an intense fluorescence
ninhydrin or fluorescamine
Protein sequences are determined using ?
Edman degradation
Edman degradation
label binds to one end of amino acid chain. Label removed along with one bound amino acid. Repeat.
edman degradation efficiency is about ?%. Reliably determines chains of ? AA’s
98%, 50
CNBr cleavage site
carboxyl side of methionine
Trypsin cleavage site
carboxyl side of lysine and arginine residues
chymotrypsin cleavage site
carboxyl side of tyrosine, tryptophan, phenylalanine, leucine and methionine
carboxypeptidase A cleavage site
amino side of C-terminal AA (not arginine, lysine, or proline)
performic acid function
breaks disulfide bonds and alkylates them to prevent reformation
locate disulfide crosslinks
separation and identification of disulfide containing peptides folowing performic acid oxidation
merrifield synthesis of peptides
t-boc amino acid reacts with DCC. Amino acid transfers to resin where it is then deprotected by CF3COOH.couple with another protected amino acid + DCC. repeat. release from resin with HF.
x-ray crystallography
xray beamed through protein crystal. Detector captures diffracted beams into images.
Common NMR protein labels
15N and 13C
NRM line caused by ?
transition between spin states
energy sepatartion between two spin states increase with ? in NMR
magnetic field strength
2D NMR reveals ? from the nuclear overhauser effect
closeness of protons in 3D structure
Three approaches to engineer a more stable T4 polymerase
reduce number of conformations of the unfolded state, stabilize the alpha-helices, increase the number of hydrophobic interactions in the core
(remove/insert) glycine to increase energy difference between folded and unfolded states
remove
(remove/insert) glycine to increase energy difference between folded and unfolded states
insert
alpha helix structure
N_H of backbone donates electrons to carbonyl of backbone four AA’s downstream in a right handed coil. A net dipole runs from the C terminal to the N terminal
qi article
PM protein complexes tagged with HPB tag containing BCCD of arabidopsis which can be captured by streptavidin beads. PM proteins tagged, RPS2, is a membrane associated disease
