-Chemical methods are important for characterizing a protein -purification is a chemical method
What aspects of a cell are used for protein purification?
-size -shape -composition -function
How do you choose a protein source?
Convenience and quantity. You can use "host cell" such as yeast to grow stuff
What are the two basic steps of protein purification
1) solubilization 2) stabilization
disrupting the cell to isolate the protein (breaking it open). can involeve: grinding, sonification, pressure, osmotic shock? Usually followed by centrifugation -membrane (not soluble) float to top
outside it's native envrionment (not stable) .... now less stable, once in unpacked environment susceptible to hydrolysis, oxidation (causing cystines to form disulfide bonds, which may be bad). often done at 4 degrees C.
Strategies for Purification
1) charge.. ion exchange, chromatography, electrophoresis, isoelectric focusing 2) Polarity... reverse phase chromotography, hydrophopbic interaction 3) Size... dialysis and ultrafiltration, gel electrophoresis, gel filtration chromotagraphy, ultracentrifugation 4) specificity...say it has a specificity for binding to something (DNA for example). Affinity chromatography.
How do you detect proteins?
1) Absorption of light... proteins absorb uv light using spectrophotometer ...use Beir's law (absorbance proportion to conc in sample) Why uv and why 280 nm? One of 20 side chains (tryptophan) heterocyclic side chain --> any protein w/ Trp can be efficiently detected w/ UV light
How do you seperate proteins out?
1) Column chromatography (color write) -used CaCo3 (chalky) making a porous fine matrix allowing molecules to travel through at different rates -ground up plant leaves
Ion Exchange Chromotography
-use ion or chromotography media (little charged beads) -if neg column, neg charged proteins will fall fastest -pos proteins stick, use ELUTION to get them off. this is done by raising pH (changing NH3 to NH2) or adding salt (much gentler). Salt gets in way of protein and beads that outcompete protein
Gel filtration, aka size exclusion chromotography
porous polymer beads slow down little proteins. big proteins go around and win
Protein is isolated by a columnn containing a ligand. ELUTION (removal) of protein is achieved w/ a high conc of ligand
How do you determine which protein you have?
Measure "activity" - anything you can measure accurately and reproducable specific activity (units /mg)
Electrophoresis (method of detection, or visualize)
Seperation on the basis of charge or size. Charge: Native gel Size: SDS, binds to hydrophobic amino acids and unfolds protein. coats protein w/ uniform mass / charge ratio (neg from sulfate)
PAGE polyacrylamide gel electrophoresis (method of detection, or visualiza)
if you SDS (all neg) smaller ones move fastest can compare to standard and determine its size
P.I. average charge (tells whether neg. or pos. charge) proteins hit isoelectric points and stop moving.
What can be found from protein sequencing?
Initially told us proteins w/ same function from different organism do not ave identical sequences.
differences arise from point mutations
some show large differences (function is not crucial)
no change (or very little) show where important part of protein sequences are
What is the value of Protein sequencing?
They tell us powerful insights into diseases and protein function
What is the overall strategy of chemical protein sequencing?
1) seperation of Chains, S-S reduction 2) Amino acid composition analysis 3) N and C terminal analysies 4) cleavage of protein into fragments 5) Sequencing of fragments 6) Overlap of fragments, assembly of the complete sequence 7) I.D. of the location (if any) of disulfide bonds
What's Edman degredation, Protein Sequencing
Key: gentle hydrolysis of N-terminal residue (unit of the polymer) reducing one amino acid at a time.
hydrolyzed N-terminal amino acid id'd by chromotography.
- Mass spectrometry is what researchers use today - Led to 2002 Nobel Prize... the trick was electrospray ionization - printout is a mass / charge ratio. -all molecules recieve the same push -use an algebra equation to solve for mass
What does a tandem mass spec do?
sequences a protein to id fragments of unique mass. -MS 1 seperates protein fragments -The collision cell results in breakage of the peptide bond -MS2 reads out the protein fragments -can tell by mass, what three amino acids made that masses -once fragments are determined, they can be back-converted into corresponding DNA sequence
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