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TERM 1
affinity chromatography
DEFINITION 1 one molecule binding to another molecule in a sample. The beads has immobilized molecule that binds with high specificity to a single protein or small number of similar proteins, nucleic acids, or other molecules TERM 2
gene fusion
technique
DEFINITION 2 gene encoding protein of interest is fused to a second gene encoding a protein that is easily purified by affinity chromatography. The second protein is called the tag. TERM 3
metal-chelate chromatography
DEFINITION 3 immobilized metal affinity chromatography IMAC- this developed because researches notice that proteins containing stretches of histidines would bind metal ions. Nickel or cobalt are loaded onto column and proteins with histidine tags are passed over the column TERM 4
tandem affinity assays
DEFINITION 4 advanced purification techniques that use multiple tags TERM 5
ammonium sulfate
cut
DEFINITION 5 another purification technique besides SEC, IEX, and affinity chromatography. It is the most basic. Proteins are generally unstable when not surrounded by water and can denature, but this uses a structure forming salt to stabilize proteins when no water is present.
TERM 6
chaotropic salts
DEFINITION 6 opposite of structure forming salts and include urea, guanidine, and HCl. They decrease the amount of teriary structure in a protein and reduce it to its linear state. It is used it a protein has become denatured into inclusion bodies. They will reduce the denatured protein to its linear form and can be slowly removed with a gradient on a column or through dialysis and the proteins will refold. Used as last resort TERM 7
Hydrophobic interaction chromatography
(HIC)
DEFINITION 7 used when there are available hydrophobic amino acids on a protein's surface which allow it to interact with a hydrophobic column. Method is like IEX but no gradient, instead the sample is placed in a buffer that is highly polar (high salt concentrations to enhance hydrophobic interactions) and is loaded onto the column. Non bounded proteins will be washed off of column and bound proteins are eluted by reducing salt conc. The least hydrophobic proteins elute first an the most are released at lower conc. of salt. TERM 8
reverse- phase chromatography
DEFINITION 8 similar to HIC and used on small molecules like peptides that are denatured and loaded onto a column containing long- chain hydrocarbons (c8-c18). Separation is based on hydrophobicity and peptides are eluted with acetonitrile or methanol in the order of their increasing hydrophobicity TERM 9
pH and solubility
DEFINITION 9 proteins can be purified by using changes in these. They are least soluble and precipitate when the pH of the solution equals the pI and when the net charge on the protein is zero. It can be used to precipitate protein of interest or its contaminants. Most helpful when pIs are different. When dialyzing a protein from on pH to another, the precipitate junk can be centrifuged out TERM 10
solvent precipitation
DEFINITION 10 used to denature or inactivate many proteins. Alcohols or acetone can be used to separate proteins in a lysate. Large volumes of organic solvents are not the first choice to purify proteins- not commonly used. If the protein survives this treatment then it can be powerful fractionation step
