Lecture 4: Protein Purification

Biochemistry 501 with Amasino/butcher at University of Wisconsin - Madison

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By: Whitney Beilke
Textbook:

Lehninger Principles of Biochemistry & eBook
Created: 2011-09-26
Size: 32 flashcards
Views: 20

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the purification protocol for purifying a protein depends on what?

the physical properties of a cell such as shape, size, composition, and function

solubilization

this involves disrupting the cell to isolate the protein, is done by gently grinding, sonification, pressure, or osmostic shock, and is generally followed by centrifugation

disadvantage of solubilization?

once the cell is broken the protein is no longer in its native environment

Stabilization

because the protein is no longer in its native environment it is less stable, therefore it usually needs to be purified at lower temperatures ( also to avoid oxidation issues)

Three strategies that use charge to purify a protein?

1. ion exchange chromatography
2. electrophoresis
3. isoelectric focusing

Two strategies that use polarity to purify a protein?

1. reverse phase chromatography
2. hydrophobic interaction chromatography

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?

affinity chromatography

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?

Tryptophan

column chromatography

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

elution

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

Affinity Chromatography

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

assay

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?

electrophoresis

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

isoelectric focusing

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?

b--hemoglobin

Cystic Fibrosis results from what mutation?

70% of cases result from a deletion of Phe508 which prevents protein folding and insertion in the membrane

edman degredation

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

mass spectrometry

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)

About this deck

By: Whitney Beilke
Textbook:

Lehninger Principles of Biochemistry & eBook
Created: 2011-09-26
Size: 32 flashcards
Views: 20

About StudyBlue

“I have been getting MUCH better grades on all my tests for school. Flash cards, notes, and quizzes are great on here. Thanks!”
Kathy