- University of Wisconsin - Madison
- Biochemistry 501
- Lecture 4
Last Modified: 2011-07-08
Related Textbooks:Lehninger Principles of Biochemistry, Fourth Edition
-purification is a chemical method
You can use "host cell" such as yeast to grow stuff
can involeve: grinding, sonification, pressure, osmotic shock?
Usually followed by centrifugation
-membrane (not soluble) float to top
.... 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.
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.
...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
-used CaCo3 (chalky) making a porous fine matrix allowing molecules to travel through at different rates
-ground up plant leaves
-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
big proteins go around and win
ELUTION (removal) of protein is achieved w/ a high conc of ligand
specific activity (units /mg)
Charge: Native gel
Size: SDS, binds to hydrophobic amino acids and unfolds protein.
coats protein w/ uniform mass / charge ratio (neg from sulfate)
can compare to standard and determine its size
proteins hit isoelectric points and stop moving.
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
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
hydrolyzed N-terminal amino acid id'd by chromotography.
- 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
-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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