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What's the first thing you have to do when you wanna study proteins?
Gotta obtain it
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All proteins come from some source, either _______ or _______.
- Biological, natural source
- Bacteria
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What do we need to do to understand how proteins function and are structured?
- Isolate them
- Sequence them
- Look at their structure
- Investigate their chemical properties
- See how they interact with each other
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About how much dry weight of a mammalian cell is taken up by proteins?
60%
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What's a proteome?
The constellation of proteins in a cell
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Understanding the function of a protein requires its _______.
Purification
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What is purification?
When a complex mixture is resolved or disconvoluted into its constituents by the successive application of physiochemical methods for separation and fractionation
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What three things are done after obtaining a single pure protein?
- Analysis of its AA sequence
- Examinaton of its functional characteristics
- Determination of its 3D structure
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What physical and chemical properties are observed during methods of protein purification in order to exploit the difference between proteins?
- Mass
- Shape
- Polarity/solubility
- Charge
- Density
- Hydrophobicity
- Ligand-binding specificity and affinity
- Special features
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What is specific activity?
The ratio of the amount of the desired protein present to the amount of total protein present in the same sample
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How is the amount of desired protein present measured?
Via its activity in our specific assay
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What three things do we need to find the specific activity of a protein?
- Assays to detect the presence of the protein of interest at each purification step
- Assays to measure TOTAL protein at each purification step
- Methods to purify the protein
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What are the different biochemical assays to detect the presence of the protein of interest?
- Colorimetric assays
- Radiochemical assays
- Spectrophometric assay
- Immunochemical assay
- Biological activity assay
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What are the different assays to measure TOTAL protein?
- Lowry method
- Bradford method
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What is the concept of a biochemical assay?
- A qualitative or quantitative method for detecting the presence and measuring the amt of a biological substance in a complex mixture of other biomolecules
- The action of an enzyme leaves a "molecular footprint"
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The action of an enzyme leaves a "____________".
Molecular footprint
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What are the two cases of a colorimetric assay?
- 1. Either the substrate or product (or both) of the reaction have a specific light absorbance that changes as the reaction progresses
- 2. There is a way to stain for substrate or product after the reaction
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What are the two cases of a radiochemical assay?
- 1. The product of the enzymatic reaction is radioactively labeled
- 2. A by-product of the enzymatic reaction is radioactively labeled
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What is spectrophotometric assay?
- The absorbance of a product or substrate changes as the reaction progresses
- The whole reaction takes place in a spectrophotometer
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What does immunochemical assay use?
Antibodies
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Give an example of colorimetric assay (case 1)
- Trying to detect the activity of the enzyme β-galactosidase (cleaving enzyme)
- Galactose is attached to a substance to make the colorless substrate galactoside (X-gal)
- When the substrate is cleaved, it turns blue
- Shows that β-galactosidase was there to cleave
- Concentration of product is assayed by absorbance at 570 nm
- Want excess substrate so amt's of product and concentration of enzyme are linear
- Map enzyme concentration vs. absorbance on a standard curve
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What's a standard curve used for?
Used as a reference
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Give an example of colorimetric assay (case 2)
- PI-Scel endonuclease (cuts DNA?)
- Stain DNA with dye that absorbs UV light and emits visible red light
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Give an example of radiochemical assay for an enzyme (case 1)
- Hexokinase (glucose phosphorylation; transfers phosphorous group from ATP to RADIOACTIVE glucose)
- Now glucose has a NEGATIVE charge
- DEAE positively charged cellulose filter paper
- ATP, ADP, and P-glucose will bind to filter; glucose will pass
- Find radioactive group stuck to filter using scintillation counter
- Quantify P-glucose
- Compare and map to standard curve
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Give an example of radiochemical assay for an enzyme (case 2)
- Theymidylate synthase (enzyme)
- Creates water through condensation
- Receives radioactive/heavy proton from substrate
- Filter through activated charcoal
- All organic molecules will bind, but radioactively labeled water will pass
- Measure activity of enzyme by assessing amt of radioactive water that filtered through charcoal
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Give an example of spectrophotometric assay for an enzyme (case 1)
- Lactate dehydrogenase (reduces NAD+ and produces NADH)
- Look for changes in absorbance from light passing through substances
- Because enzyme uses NADH as a substrate, its concentration will decrease according to enzyme concentration
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Describe immunochemical assay for protein detection (case 1)
- HIV coat proteins in human serum
- a. Standard enzyme-linked immunosorbant assay (ELISA)
- Antigen → Add specific antibody → Add enzyme-linked antibody → add substrate, get yellow
- Only get reaction if enzyme present; Enzyme only present if antibody binds
- b. Sandwich ELISA
- First antibodies → Add antigen → Add second enzyme-linked antibody → Add substrate, get yellow
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Give an example of immunochemical assay for protein detection
- p-nitrophenyl-phosphate (colorless substrate)
- Alkaline phosphatase (antibody coupled enzyme)
- p-nitrophenol (bright yellow product)
- Can also be seen as colorimetric
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What are the different biochemical ways to assay TOTAL protein in a sample?
- Lowry Method
- Bradford method
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What is Lowry method?
- Named for the biochemist Oliver H Lowry
- His 1951 paper describing the technique is most-highly cited
- Better if working with membrane potentials
- Not good in cytosol b/c sensitive to the reducing agents normally found there
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What is Bradford method?
- Developed by Marion M Bradford (1976)
- Requires very low concentrations of protein (thus samples usually have to be diluted)
- Sensitive to detergents
- Better for cytosolic proteins
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What is the biuret test?
- Chemical test for detecting the presence of peptide bonds
- In the presence of peptides, a copper2+ ion forms violet-colored coordination complexes in an alkaline soln
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Why can the biuret reaction be used to assess the concentration of proteins?
B/c peptide bonds occur with the same frequency per amino acid in the peptide
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The intensity of color (absorption at 540nm) in a biuret test is directly proportional to what?
Protein concentration
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The intensity of color in a biuret test is ________ proportional to the protein concentration.
directly
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What is directly proportional to the concentration of proteins in a biuret test?
The intensity of color
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The Lowry method for protein determination combines what two tests?
- The Biuret test (the rxn of Cu2+ ions with the peptides under alkaline conditions)
- The oxidation of aromatic protein residues using the Folin-Ciocalteu reagent
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How does the oxidation of aromatic protein residues using the Folin reagent affect sensitivity?
Increases it 100 times
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The biuret test is unique to which method for protein determination?
Lowry method
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What kind of proteins are used in the Bradford method?
Proteins with basic and aromatic side chains
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For most methods, what do you need to make to compare your unknown sample to?
A standard curve
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Every protein purification takes place in what?
A buffer
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What is a protein purification buffer?
- Aqueous mixture of components that:
- -- extract the desired protein into solution
- -- helps preserve the structure and function of the desired protein
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How does a protein purification buffer help preserve the structure and function of the desired protein?
- It resists any change in pH
- It contains other reagents that help prevent the desired protein from suffering unwanted oxidative and/or proteolytic damage
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How do protein purification buffers resist any change in pH?
It contains equimolar amounts of a conjugate acid-base pair that has a pKa value at or very near phisiological pH
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What are some typical buffer components used in protein purification?
- Buffering species
- Salt
- Reducing agent
- Chelator
- Protease inhibitor
- Other
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Why are buffering species used in buffers for protein purification?
To hold pH constant
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Why is salt used in buffers for protein purification?
- Proteins are more soluble at moderate ionic strength
- Helps maintain dielectric constant
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Why are reducing agents used in buffers for protein purification?
To prevent oxidation of Cysteine
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Why are chelators used in buffers for protein purification?
To remove heavy metal ions
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What are chelators?
Substance bonded to a metal ion at two or more points
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Why are protease inhibitors used in buffers for protein purification?
To revent proteolysis
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As purification of proteins proceeds, what is being removed?
Extraneous proteins and other contaminants
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What rises as the purification of protein proceeds?
Specific activity
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When does specific activity eventually reach a maximum?
As purification of protein proceeds till the end
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What is one measure of protein purity?
When specific activity remains constant and cannot be further increased
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What do you use to isolate proteins from specific subcellular fractions/organelles?
Differential centrifugation
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What is differential centrifugation used for?
To isolate proteins from specific subcellular fractions/organelles
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What is differential centrifugation?
- Crush/blend tissue completely
- Centrifuge at low speed and separate supernatant
- Repeat but centrifuge at higher speed than before
- Repeat until supernatant is relatively clear
- Supernatant contains soluble proteins
- What steps you take depends on your protein
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What are the steps in differential centrifugation?
- Break open the cell
- If the protein is in the cytosol, the crude lysate may be filtered or centrifuged to remove the cell debris and stuff; Protein of interest is in the supernatant
- If the protein is in a membrane or organelle, repeated centrifugation and isolation of pellet or supernatant will provide access to the organelle or membrane
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How would you break open an animal cell for differential centrifugation?
Place cell in hypotonic/osmotic solution
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How would you break open a plant/bacteral cell for differential centrifugation?
- They have cell walls
- Blender
- Sonicator (ultrasound)
- French press
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What steps do you have to take for differential centrifugation if the protein is in the cytosol?
- After cells have been broken, the crude lysate may be filtered or centrifuged to remove the cell debris
- The protein of interest is in the supernatant
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What steps do you have to take for differential centrifugation if the protein is in a membrane or organelle?
Repeated centrifugation and isolation of the pellet or supernatant will provide access to the organelle or membrane
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How do we get the protein from the supernatant?
- We need to know something about the protein
- Some proteins will precipitate if we add salt (usually ammonium sulfate) → "Salting out"
- If we know the approximate size of the protein, we can use a dialysis process with a membrane of the right size
- Column chromatography can separate proteins according to charge, size, affinity for a specific ligand, etc.
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What is salting out?
- After proteins solubilized, they can be purified based on solubility
- Use ammonium sulfate (NH4SO4) as salt
- Takes away water by interacting with it
- Makes protein less soluble b/c hydrophobic interactions among proteins increases
- Aggregation of proteins by their most hydrophobic surfaces b/c no tenough free H2O for solvation
- Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments)
- One fraction has the protein of interest
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Regardless of the specific type of chromatography, What should you always do to get a protein from supernatant?
- Start with a sample of our protein in soluble form
- Run it through a column
- Collect fractons at different times
- Use a biochemical assay to identify the fraction containing our protein
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What are the two phases of columns in column chromatography?
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What is a stationary phase in column chromatography?
- Solid porous matrix
- Protein samples go through and interact with this phase
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What is a mobile phase in column chromatography?
- Protein sample
- Flows over the stationary phase
- Carries along with it the sample to be separated
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What are the steps of column chromatography?
- Start with a sample of our protein in soluble form
- Use a buffer/soln to "push" our protein (mobile phase running down column)
- Proteins will be separated as they will run at different speeds depending on their properties
- Collect fractions of eluate at different times
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What is effluent?
What you collect during column chromatography
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What is size exclusion chromatography?
- Gel filtration
- Proteins are separated by size
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What is the process for size-exclusion chromatography/gel filtration?
- Column contains a cross-linked polymer
- Polymer contains small pores
- Proteins that are small enough to enter the pores will be stuck and delayed
- Larger molecules pass more freely; apprearing on the earlier fractions
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What is ion-exchange chromatography?
Separates protein according to charge
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What is the process for ion-exchange chromatography?
- Column contains cation or anion exchangers (polymers w/ charged functional groups)
- Proteins move through the column at rates determined by their net charge at the pH used (and elution buffer used)
- With cation exchangers, proteins with a more negative net charge move faster and elute earlier as they are repelled
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What is affinity chromatography?
Based on ligand binding affinity
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What are the first steps of affinity chromatography?
- Beads have a ligand covalently attached that will bind the protein of interest and retard its migration through the matrix
- Unwanted species will wash through the column
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What are the second steps of affinity chromatography?
- After unwanted proteins have washed
- A solution containing the same ligand is now added to the column (could also be high salt or different pH to disrupt interactions)
- The protein is eluted by the ligand solution
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What does eluted mean?
Remove by washing withsolvent
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What is hydrophobic interaction chromatography (HIC)?
- Relatively new technique
- Takes advantage of the fact that most proteins have hydrophobic pockets and of the hydrophobic effect
- Matrix of the column consists of hydrophobic ligands
- Protein solution is mixed with high levels of salt, promoting hydrophobic effect (but not too high b/c can cause ppt)
- Proteins will elute according to their hydrophobicity, with the most hydrophobic proteins beind delayed the most
- Elution is done with solutions of DECREASING salt concentration
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What happens if the salt levels are too high in the protein solution during HIC?
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How are chromatographic methods typically enhanced?
Using high performance liquid chromatography
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What is high performance liquid chromatography?
- HPLC
- Uses high pressure pumps that speed up mvmt of the protein molc down the column
- Speical columns that can sustain the pressure of the pressurized flow (no compressible resin and strong columns)
- Results in reduced transit time which limits the diffusion of the protein and enhances the resolution
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What does HPLC result in?
- Reduced transit time
- Limits the diffusion of protein
- Enhances resolution
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When is electrophoresis used?
For protein analysis
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How is separation in analytical scale commonly achieved?
By electrophoresis
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What is electrophoresis?
- Electric field pulling on proteins according to their charge
- Gel matrix hinders mobility of proteins according to their size and shape
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Why is electrophoresis useful?
- Allows us to visually separate proteins and assess the degree of purity of a mixture
- Can also determine the MW and pI of proteins
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What is polyacrylamide gel electrophoresis?
- Different samples are loaded in wells or depressions at the top of the polyacrylamide gel
- The proteins move into the gel when an electric field is applied
- The gel minimizes protein mvmts other than those induced by the electric field
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The migration of proteins in a gel is a function of what?
Their size and shape
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How can proteins be run in gels?
- In their native form
- Bound to sodium dodecyl sulfate (SDS)
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How does SDS allow proteins to run in gels?
- Denatures the protein (no 3D structure interference)
- Makes the protein negatively charged with overall charge being proportional to the length of the protein
- Rate of mvmt will only depend on size, with smaller polypeptides migrating more rapidly
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When SDS binds to proteins, why is the overall charge proportional to the length of the protein?
Proteins bind approximately 1 molecule of SDS/AA
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What must be mixed with proteins to run a gel?
Gel Loading Buffer
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Why must a gel loading buffer be mixed with proteins to run a gel?
- It has glycerol (density)
- It has bromophenol blue (dye)
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Why is glycerol in gel loading buffer useful to run a gel?
- Density
- Need the protein to stay at bottom of well/groove until it's time for electricity
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Why is bromophenol blue in gel loading buffer useful to run a gel?
- It's a dye that can be seen by the naked eye
- Used to monitor the migration of proteins in the gel
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What is a ladder?
Has proteins of different known sizes
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Why are ladders used when running a gel?
Used to estimate the size of the protein
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What are the final steps of SDS-PAGE?
- Once the gel has run, it's placed in a Comassie Blue soln that binds to proteins
- Each band in the gel represents a different protein or protein subunit
- Can see different purification steps
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SDS-PAGE can be used to estimate what about the protein?
The molecular weight
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What is isoelectric focusing?
A procedure used to determine the isoelectric point of a protein
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How is the pI of a protein determined using isoelectric focusing?
- A pH gradient is established in a gel
- When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI
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What is two-dimensional electrophoresis?
- Isoelectric focusing and SDS-PAGE are combined
- Proteins are first separated by isoelectric focusing in a thin strip gel
- The gel is tehn laid horizonally on a second, slab-shaped gel (SDS-PAGE)
- The proteins are separated by SDS polyacrylamide gel electrophoresis
- Thus, the original protein mixture is spread in two dimensions
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What does horizontal separation show in 2D electrophoresis?
Reflects differences in pI
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What does vertical separation show in 2D electrophoresis?
Reflects differences in molecular weight
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What important things can be compared using 2D gels?
- Protein pools
- (treatment, diseases, etc.)
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