Four strategies that use size to purify a protein?
1. dialysis and ultrafiltration 2. gel electrophoresis 3. gel filtration chromatography 4. ultracentriguation
A strategy that uses specificity to purify a protein?
how is a spectrophotometer used to identify proteins?
light goes through a monochomater and the sample to a detector that measure the amount of light that was absorbed, absorption is proportional to the concentration of protein present
which amino acid absorbs UV light more efficiently than the others?
separates pigments through a column of calcium carbonate, separates based on size
ion exchange chromatography
column has a negative charge, the positive charged proteins stick to the negatively charged beads, therefore the proteins that drop out of the bottom of the column first are the negative ones
How is elution achieved in ion exchange chromatography?
achieved by changing the pH or salt conditions, match the pH so it is neutral, then there is no charge and the protein can be removed
removal of a protein out of a column
Gel filtration/Size Exclusion Chromatography
porous beads are in a column and act as a "molecular sieve," the smaller molecules are get stuck in the pores and take longer to travel through the column, therefore the large proteins are the first to travel through the column
protein is isolated by a column that contains a ligand, for example ATP binding proteins can be isolated with a column that contains bound ATP, elution occurs with increased concentration of free ATP
reproducible test to discover a function (should be quantitative)
as the protein is purified what happens to its activity? its specific activity?
its activity goes down, its specific activity goes up
what method of protein purification revolutionized science?
separation on the basis of charge by application of an electric field, can separate on the basis of charge or size
SDS gel electrophoresis
separates on the basis of size, the protein is denatures in the presence of an anionic detergent (sodium dodecyl sulfate) that binds strongly to the protein
how does the polyacrylamide gel function?
it's a porous gel that allows the smaller proteins to pass but the larger ones are stuck near the top and travel a smaller distance, then the stock gel is dyed to see how far the proteins traveled
can chromatography be used to identify a protein?
no! it is only a separation technique, not a detection technique, electrophoresis can be used to identify a protein
uses a gel solution with a pH gradient (the pH changes down the length of the test tube), the molecules with low PI's will go down toward the lower pH to the positive end until the neutral state is achieved
what do we do with a protein after purification?
it can be sequenced
sickle cell anemia results from a mutation in what?
Cystic Fibrosis results from what mutation?
70% of cases result from a deletion of Phe508 which prevents protein folding and insertion in the membrane
is a chemical method for protein sequencing, hydrolyzing N-terminal residue to remove one amino acid at a time, the hydrolyzed N-terminal amino acid is identified by chromatography, 30 amino acids can be sequenced from a peptide with this method
glass capillary ionizes the protein (surrounds it with positive charges), the protein is sprayed into a vacuum as a gas phase (need capillary to vaporize this and increase the voltage), the heavier protein won't fly as far (distance is a function of its mass)
disadvantage of mass spectrometry
it's a sensitive technizue, need small amounts of protein
what does the peak in mass spectrometry indicate?
mass of the uncharged protein
Tandem Mass Spectrometry
uses 2 spectrometers to break the protein down even more into fragments of unique mass, tells you the composition of a small protein sequence but doesn't tell you the order (use a computer for this)
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