BCHS 3304 Exam 2 Questions and CORRECT
Answers
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.
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
Ion exchange chromatography
Most frequently used anion exchanger
Diethylaminoethyl (DEAE)
Most frequently used cation exchanger
,Carboxymethyl (CM)
Separation based on solubility differences between phases
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
Cation Exchange
Negative
Protein charge when pI < pH
Positive
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
Anion Exchange
, - UV absorbency
- Fluorescence
- Radioactivity
- Staining with dyes (Ninhydrin, Iodine, Sulfuric acid)
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
Gel Filtration/Size Exclusion Chromatography
Vt = Vx + Vo
Vo = Void volume
Vx = occupieid by gel beads
Answers
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.
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
Ion exchange chromatography
Most frequently used anion exchanger
Diethylaminoethyl (DEAE)
Most frequently used cation exchanger
,Carboxymethyl (CM)
Separation based on solubility differences between phases
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
Cation Exchange
Negative
Protein charge when pI < pH
Positive
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
Anion Exchange
, - UV absorbency
- Fluorescence
- Radioactivity
- Staining with dyes (Ninhydrin, Iodine, Sulfuric acid)
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
Gel Filtration/Size Exclusion Chromatography
Vt = Vx + Vo
Vo = Void volume
Vx = occupieid by gel beads
