module 3 Flashcards
proteome definition
total protein content in a cell
types of proteomics (study of proteins)
structural - analyzes structure of proteins being produced expressional - considers protein expression under certain conditions
interaction - how proteins interact with other molecules
protein purification and its steps
isolating proteins from their source
steps: 1. cell fractionation 2. series of centrifugation using centripetal acceleration
dialysis
protein solution is put into a bag, bathed in a buffer solution. Large molecules remain and small molecules go through pores.
Solvents, ions and buffer can diffuse easily across the semipermeable membrane, but larger molecules are unable to pass through the pores.
salting out
purification technique for proteins based on differential solubility in salt solutions, causes protein to compete with salt. The protein is precipitated and less water molecules are able to interact with the protein. Causes proteins to form hydrophobic bonds with each other.
Involves ammonium sulfate and hydrophobic amino acids on exterior interaction with water.
Effect of salting out
salt in small concentrations causes ammonium and sulfate to be separated. When concentration of salt is increased, protein solubility increases and ceases to interact with water.
Salting out for different protein molecules
Different protein molecules precipitate at different concentrations of salt solution allowing for some proteins to precipitate while others interact with the water.
Column chromatography
includes stationary phase, which retards the flow of the sample and the mobile phase, where the mixture to be separated moves
gel filtration chromatography
separates biomolecules on basis of size, smaller proteins move faster than larger proteins
non porous vs porous resins
non porous proteins move faster
affinity chromatography
powerful column separation procedure based on specific binding of molecules to ligands
proteases
enzymes that typically break peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis .
SDS function
reduces proteins so they assume a rod like shape, useful for gel electrophoresis
merceptoethanol
sulfhydryl-containing reagent that becomes oxidized as they reduce disulfide bonds in other molecules
isoelectric focusing
pH gradient established in tube containing an acrylamide gel matrix. An electric field is applied and proteins move according to their charge. When molecules reach their pI, they stop moving.
2D gel electrophoresis
SDS page and isoelectric focusing are combined for proteomics, allowing all proteins of a cell/tissue to be studied simultaneously.
First step is isoelectric focusing, second step is use of SDS, which coats proteins with negative charge and denatures them, allowing SDS page to separate proteins exclusively according to molecular weight .I
Histidine properties
flexible proteins has certain affinity to different ions, allowing for it to be used as a tag in recombinant proteins
His-tags are often used to identify expressing cells when producing recombinant proteins.
ion exchange chromatography
separates substances on the basis of charge
involves cation exchange media (negatively charged groups on stationary phase trying to slow positive molecules) and anion exchange media (positively charged stationary phase trapping negative molecules)
What is true about SDS page?
smaller proteins move faster.
HPLC (high phase liquid chromatography)
chromatography technique that gives fast and clean purifications
Electrophoresis definition
separates molecules on the basis of ratio of charge to size
What do specific activity of enzyme, fold purification, and total activity mean in regard to protein purification
Total activity always decreases during purification. When a protein is purified, the specific activity will increase as a product of the fold purification. If the total activity is decreased too much and fold purification is not that high, the step is most likely not needed.
Strategy to detect primary structure of protein
several samples of interest are prepared to identify individual amino acids, identify the N terminal amino acid, deduce the sequence of smaller peptides, and combine info from overlapping peptides used.
trypsin
specific to Arg/Lys, proteolytic enzyme
charged side chain ends up at C terminal end of peptides
