Biochemistry- Protein Purification – Flashcards

1. For medical use 2. To study structure and function 3. For use as a reagent in the lab

- proteins are present in cells or secretions and are therefore complex mixtures; plus other DNA, lipid, small molecules, etc. (need to remove unwanted material- "contaminants")

1. Size and Shape 2. Charge 3. Location 4. Surface Hydrophobicity

1. Need an assay method - must be able to detect in a mixture - measure some property of the protein of interest 2. Decide on starting material - human tissues, plants, bacteria, etc.

- both are used to purify protein - start with preparative method, follow with analytical

Homogenizes ("burst up") material - large scale - milligrams to kilograms - divide mixture into "fractions"

Detects specific protein of interest - small scale - micrograms or less - small samples of fractions for analysis

break open tissue or cells to release protein - sonication= high frequency sound waves - grinders - blenders/homogenizers - pressure (to force things out) - detergents - lysozyme (get more protein out) - compatible buffer solution (one in which the protein is "native") --> must be gentle, want to be able to make it back into a functional protein

- spin homogenate to separate by size/density - "Differential Centrifugation" - low speed- 100 to 1000 xg (force of gravity) (whole cells, nuclei, large defiris are pelleted - medium speed- 2000 to 50000 xg (mitochondria, lysosomes) - high speed- 60000+ xg (ribosomes, viruses, large macromolecules)

smaller and less dense components

larger and more dense components

- further separates proteins based on characteristics like charge and size

1. Mobile Phase- aqueous buffer solution 2. Solid Phase- usually small beads (matrix held in a cylinder or "column" - proteins in a mobile phase interact (or not) with the matrix - continuous flow of buffer solution carries unbound proteins along

1. support goes into a column 2. equilibrium 3. sample binding 4. column wash (to wash away impurities)- same as buffer used for equilibration 5. sample elution (wash support with appropriate buffer) 6. column regeneration (prepares column for further use and storage) - chromatography can be performed in different types of vessels at different pressures (simplest form of chromatography is carried out in a beaker)

if single buffer is used

if multiple buffers are used

- eluting proteins off the columns - the effluent is collected (eluted) in fractions - generally, a constant flow rate is maintained, and a fraction is collected every few minutes (rate depends on how big the column is) - usually get many fractions from a single run - small samples from each fraction are used to detect specific protein of interest and to measure total protein (must do work to see which fractions have proteins in it and which don't)

absorbance at 280 nm

absorbance at 280 nm

1. Ion-exchange chromatography 2. Gel-filtration chromatography 3. Affinity chromatography (see note for processes)

(1) Ion exchange relies on attraction of opposite charges e.g. DEAE is positively charged --> binds negative ions (anions) --> if a protein is negatively charged, it will bind Exchanging with ions previously bound - to get protein off, increase salt concentration - e.g. increase concentration of anions such as Cl- to compete - usually done as a gradient of increasing concentration (first weak ones will come off and as you increase concentration of salt, the stronger ones will come off) - small proteins never come off first (2) Charge on proteins varies with pH

the pH where a protein's net charge=0 - pH>PI=negative net charge - pH<PI=positive net charge

- beads contain holes - if a protein can get into the holes, it is slowed down - if a protein is excluded, it will come out early - another name is "size exclusion" - in addition to size, protein shape also affects elution speed (bigger takes longer)

- relies on a specific interaction between a protein and a ligand - "ligand" = a compound that binds to a protein - if a ligand is attached to a column matrix, certain proteins will bind to it, others won't - flow of buffer will wash away unbound proteins, leaving only the specifically bound protein - most powerful form of chromatography but requires knowledge of what ligand to use

1. Adding more ligand or 2. Changing the pH of the salt concentration

- tells you if your protein is pure - most common analytical method - SDS=sodium dodecyl sulfate (an ionic detergent) - separates on the basis of polypeptide chain size - charged molecules migrate in an electric field (negative on top, positive on bottom) - get charged by SDS, so all become negative - a gel acts as a molecular sieve (small molecules go faster) - hydrophilic polymer in long chains - lower polyacrylamide=bigger holes=more proteins can get through (100kda=big, 20kda=small) - gels can be single concentrations (eg. 6% or 12%) or a gradient (6-15%) - low % gels- used for high MW proteins - high % gels- used for low MW proteins - gradients used when you want to examine a range of different sized proteins - is used to determine the molecular weight of a subunit of a purified protein - involves denaturation of proteins in the presence of detergents (sodium deodecyl sulfate) - is commonly used to monitor protein purification procedures - is commonly used in a technique known as Western blot - the chains move due to charge - it separates based on size (see note for diagram/process)

- in SDS-PAGE, a gel is made of polyacrylamide (6%-15%)

Denatures proteins and gives it a negative charge

1. X-ray crystallography 2. Protein activity 3. Kinetic studies 4. Ligand binding assays