BCHS 3304 Exam 2 || Errorless Answers 100%.
Protein precipitation caused by an increase in the salt concentration.
As more salt is added ,the solubility of the protein decreases (particularly with sulfate salts).
- Result of competition between added salt ions and other dissolved solutes for molecules of
solvent
- At high salt concentrations, so many of the added ions are solvated that there is significantly
less bulk solvent available to dissolve other substances, including proteins.
- Ammonium sulfate is most commonly used reagent. correct answers Salting Out
- Separates anions and cations
- Charged molecules bind to oppositely charged groups that are chemically linked to a matrix
such as cellulose or agarose correct answers Ion exchange chromatography
Most frequently used anion exchanger correct answers Diethylaminoethyl (DEAE)
Most frequently used cation exchanger correct answers Carboxymethyl (CM)
Separation based on solubility differences between phases correct answers Adsorption
chromatography
A process in which cations bind to anionic groups on cationic exchangers.
- Proteins with different pI values will have varying degrees of charge at a given pH and different
affinities for negatively charged surface groups on the particles of cation exchange media.
- This facilitates separation correct answers Cation Exchange
,Negative correct answers Protein charge when pI < pH
Positive correct answers Protein charge when pI > pH
A process in which anions bind to cationic groups on anion exchangers.
- Anion exchange resins will bind to negatively charged molecules as they are positively charged.
- Commonly used to purify proteins, AAs, sugars/carbs, and other acidic substances with a
negative charge at higher pH levels correct answers Anion Exchange
- UV absorbency
- Fluorescence
- Radioactivity
- Staining with dyes (Ninhydrin, Iodine, Sulfuric acid) correct answers Materials in paper
chromatography can be visualized by :
Separates proteins based on size
- Stationary phase consists of gel beads containing pores
- Gel bead consists of a gel matrix enclosing an internal solvent space
- Aqueous solution of molecules passed through a column containing the beads and pores
- Molecules that are too large to pass through the pores are excluded from the solvent volume
inside the gel beads
- Smaller molecules are included in the gel as they pass through the pores
- Smaller molecules consequently migrate through the column more slowly than the large
molecules that are excluded from the gel
- Large molecules elute first
, - Can be used to reduce salt concentration of a protein solution correct answers Gel
Filtration/Size Exclusion Chromatography
Vt = Vx + Vo
Vo = Void volume
Vx = occupieid by gel beads
Ve = elution volume
Before swelling, dry bead size ~5% of Vt
60% of Vt is holes correct answers Gel Filtration Equation
Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of
interest
- Based on molecular complementarity between an enzyme and substrate
- A ligand that specifically binds the protein of interest is covalently attached to an inert matrix
- Only a protein that binds to this ligand will "stick" to the column
- When impure protein is passed through chromatographic material, desired protein binds to
immobilized ligand
- Other substances are washed through the column with the buffer
- Desired protein can be recovered in highly purified form by changing the elution conditions to
release the protein from the matrix
- Advantage is the ability to exploit a protein's biochemical properties rather than physiochemical
properties between proteins as exploited by other methods of chromatography
- Greater power of separation correct answers Affinity Chromatography
Protein precipitation caused by an increase in the salt concentration.
As more salt is added ,the solubility of the protein decreases (particularly with sulfate salts).
- Result of competition between added salt ions and other dissolved solutes for molecules of
solvent
- At high salt concentrations, so many of the added ions are solvated that there is significantly
less bulk solvent available to dissolve other substances, including proteins.
- Ammonium sulfate is most commonly used reagent. correct answers Salting Out
- Separates anions and cations
- Charged molecules bind to oppositely charged groups that are chemically linked to a matrix
such as cellulose or agarose correct answers Ion exchange chromatography
Most frequently used anion exchanger correct answers Diethylaminoethyl (DEAE)
Most frequently used cation exchanger correct answers Carboxymethyl (CM)
Separation based on solubility differences between phases correct answers Adsorption
chromatography
A process in which cations bind to anionic groups on cationic exchangers.
- Proteins with different pI values will have varying degrees of charge at a given pH and different
affinities for negatively charged surface groups on the particles of cation exchange media.
- This facilitates separation correct answers Cation Exchange
,Negative correct answers Protein charge when pI < pH
Positive correct answers Protein charge when pI > pH
A process in which anions bind to cationic groups on anion exchangers.
- Anion exchange resins will bind to negatively charged molecules as they are positively charged.
- Commonly used to purify proteins, AAs, sugars/carbs, and other acidic substances with a
negative charge at higher pH levels correct answers Anion Exchange
- UV absorbency
- Fluorescence
- Radioactivity
- Staining with dyes (Ninhydrin, Iodine, Sulfuric acid) correct answers Materials in paper
chromatography can be visualized by :
Separates proteins based on size
- Stationary phase consists of gel beads containing pores
- Gel bead consists of a gel matrix enclosing an internal solvent space
- Aqueous solution of molecules passed through a column containing the beads and pores
- Molecules that are too large to pass through the pores are excluded from the solvent volume
inside the gel beads
- Smaller molecules are included in the gel as they pass through the pores
- Smaller molecules consequently migrate through the column more slowly than the large
molecules that are excluded from the gel
- Large molecules elute first
, - Can be used to reduce salt concentration of a protein solution correct answers Gel
Filtration/Size Exclusion Chromatography
Vt = Vx + Vo
Vo = Void volume
Vx = occupieid by gel beads
Ve = elution volume
Before swelling, dry bead size ~5% of Vt
60% of Vt is holes correct answers Gel Filtration Equation
Uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of
interest
- Based on molecular complementarity between an enzyme and substrate
- A ligand that specifically binds the protein of interest is covalently attached to an inert matrix
- Only a protein that binds to this ligand will "stick" to the column
- When impure protein is passed through chromatographic material, desired protein binds to
immobilized ligand
- Other substances are washed through the column with the buffer
- Desired protein can be recovered in highly purified form by changing the elution conditions to
release the protein from the matrix
- Advantage is the ability to exploit a protein's biochemical properties rather than physiochemical
properties between proteins as exploited by other methods of chromatography
- Greater power of separation correct answers Affinity Chromatography
