Biochem Ch 5-Protein Purification
About this set
Created by:
meredithredick on July 27, 2011
Subjects:
Log in to favorite or report as inappropriate.
Order by
36 terms
Terms | Definitions |
|---|---|
 Steps of Protein Isolation | 1. selection of protein source 2. solubilization 3. stabilization 4. assay |
| Protein Source Selection | -different variants of a given protein may occur in diff. tissues or even in diff. compartments of same cell -tissue from domesticated animals, e.coli, or yeast tends to be easier to obtain. -molecular cloning |
| molecular cloning | isolate a protein-producing gene, genetically engineer it, and overexpress it in e.coli so that you get lots of it |
| Solubilization of Protein (liberation from cell) into crude lysate | a. Lysis or mechanical disruption b. filtering out of cellular debris (for some, differential centrifugation is effective, though detergents or organic solvents may be necessary if the protein is tightly bound to a lipid membrane) |
| Lysis | For cytosolic proteins-osmotic lysis (hypotonic) -if has cell wall, lysozyme or chemical degradant (eg acetone, toluene, although these may denature the protein) is necessary to degrade cell wall |
| Mechanical Cell Disruption | blending/grinding cells (using homogenizer, French press, sonification (ultrasonic vibrations)) to open them up |
| stabilization of proteins | necessary because externalized proteins are very vulnerable control: -pH (buffer) -temperature (usually 0C is safe, but some proteins require <-100C) -if you know thermal stability, can manipulate temps to denature + precipitate contaminant proteins -must maintain pH and temp unfavorable to degradative enzymes -minimize frothing other sensitive factors: -oxidation of cysteine -heavy metal contaminants may bind tightly and irreversibly -[salt] -polarity of solution -microbial populations |
| cold-labile proteins | proteins that don't tolerate cold well |
| minimize frothing | -exposure to air/water interface denatures -adsorption to surfaces if low concentration can cause loss of protein |
| Assay of Proteins-quantitatively determine presence | types: -fluorescence measurements or spectra -acid-base titrations -coupled enzymatic rxns |
| assay of non-enzyme proteins | -specific binding of radioactive molecules -hormone effects on a standard tissue -antibody -radioimmunoassay (eg ELISA) |
| ELISA | "enzyme-linked immunosorbent assay" 1. effective antibody is immobilized on, eg, polystyrene 2. lysate applied to surface 3. resulting A-P complex is reacted with a second protein-specific antibody to which an easily assayed protein has been enzymatically linked 4. assay! |
| Charge-based Separation Procedures | -ion exchange chrom. -electrophoresis -isoelectric focusing |
| polarity-based separation procedures | -adsorption chrom. -paper chrom. -reverse-phase chrom. -hydrophobic interaction chrom. |
| size-based separation procedures | -dialysis +ultrafiltration -gel electrophoresis -gel filtration chrom. -ultracentrifugation |
| specificity | affinity chromatography |
| What affects the solubility of a protein? | 1. salt concentration 2. type of solvent 3. pH |
| effects of salt concentration on solubility | -at low ionic strength the solubility increases with [salt]--"salting in"--because additional counterions shield the protein molecules' multiple ionic charge and therefore increase solubility -at high ionic strength, solubility decreases--salting out |
| ionic strength | I = 0.5 (sum of ion concentration and (ionic charge)^2) essentially a measure of ion concentration |
| salting out | at high ionic strength, solubility decreases with increase in salt because solvent is so busy dissolving ions that it doesn't have enough molecules to solvate the protein. -by adjusting [salt] to just below the precip. pt of the protein, unwanted proteins can be precipitated out.then you can precipitate out the desired protein and isolate it. |
| salting in | at low ionic strength solubility increases with increase in salt -is easier at a pH far from isoelectric point to increase electrostatic forces between protein and solution |
| ammonium sulfate | good salting out reagent |
| ions that increase protein solubility rather than facilitating salting out | I- ClO4- SCN- Li+ Mg+2 Ca+2 Ba+2 -these also tend to denature proteins (ions that decrease solubilities tend to stabilize the proteins) |
| Effects of Organic Solvents on solubility | solvents with low dielectric constants that are water-miscible, like ethanol and acetone, are good protein precipitants bc the solution cannot easily dissolve charged particles |
| dielectric constant | measure of how easily charge dissipates in the solution -low dielectric constant magnifies differences in salting out behavior of proteins |
| effects of pH on solubility | - as the protein's net charge increases, it should be increasingly subject to salting in because of increased electrostatic interactions, which cause precipitation and aggregation -in solutions of moderate [salt], solubility will be a minimum at protein's pI and increase about this point wrt pH (can use this info to precipitate by adjusting pH to pI) |
| isoelectric point (pI) | -the pH at which the positive charges on the molecule exactly balance its negative charges -means molecule is immobile in electric field |
| crystallization | generally tricky |
| chromatographic separation | -mobile phase is percolated through a column of porous solid matrix ("stationary phase") -applied in a continuous fashion so that eventual segregation occurs "gas-liquid chrom" means gas mobile (first word) and liquid stationary (second word) |
| ion exchange | ions are electrostatically bound to an insoluble "exchanger"-these ions are reversibly replaced by ions in solution -polyelectrolytes (polyionic polymers) such as proteins can bind both anionic and cationic exchangers -proteins with low affinity for ion exchangers move more quickly through column |
| protein's affinity for ion exchanger | -depends on other ions in solution -affinity is also pH-dependent bc molecule's net charge varies |
| stepwise elution | tightly bound proteins that don't move far can be further separated via stepwise changes in salt and pH |
| gradient elution | continuously varied salt concentration/pH to sequentially release various proteins (eg linear gradient) |
| types of resins (support matrices) | polydextran gels cross-linked polyacrylamide gels cellulose silica/glass (modern) |
| characteristics of gels | -polydextran is good bc highly substituted charged groups, so have higher loading capacity than cellulose -both PAA and polydextran are easily compressed by high pressure, decreasing overall eluant flow -noncompressible matrices of silica and glass are more common now |
| Paper Chromatography | ... |
First Time Here?
Welcome to Quizlet, a fun, free place to study. Try these flashcards, find others to study, or make your own.