43 Questions
What is the function of the stationary phase in ion exchange chromatography?
To separate molecules based on charge or polarity
What type of molecule does an anion exchange resin bind to?
Anions
When is a cation exchange resin most likely to move slowly down the column in ion exchange chromatography?
When positively charged molecules are added
What type of molecules is ion exchange chromatography generally used as a purification technique for?
Peptides, nucleotides, DNA, and proteins
What is the net charge of a molecule?
The overall charge on the molecule when all individual charges are added together
In what situation would an anion exchange resin move quickly down the column in ion exchange chromatography?
When positively charged molecules are added
What is the characteristic of strong exchangers in ion exchange chromatography?
They are always ionized
What type of molecule does a cation exchange resin bind to?
Cations
What is the purpose of an indirect ELISA?
To detect and quantify specific proteins in a complex mixture
In sandwich ELISA, each well of the plate is coated with:
Antibody
What is one of the disadvantages of ELISA?
Time-consuming
What happens if the sample in indirect ELISA contains antibodies to the antigen?
It binds to the antibodies in the well
What type of ELISA is used for quantitating the amount of an antigen in a sample?
Sandwich ELISA
What is added after washing the wells in sandwich ELISA?
Different antibody conjugated to an enzyme
What does the enzyme substrate turn if antigen is present in sandwich ELISA?
Yellow
What is used for HIV testing?
Indirect ELISA
What does ELISA primarily offer in terms of advantages?
Specificity and sensitivity
In ion exchange chromatography, what can be altered to elute molecules from the column?
pH or salt concentration
What is the function of a pH gradient in protein separation using isoelectric focusing?
Separating molecules based on charge
What is the main principle behind reverse phase chromatography?
Using a polar stationary phase and non-polar mobile phase
What is the main advantage of TLC (thin layer chromatography)?
Visualizing components and cheap
What is the distinguishing feature of affinity chromatography?
Separates based on affinity for a specific ligand
What is the purpose of western blotting in conjunction with SDS-PAGE?
Visualizing specific proteins using an antibody
What can be used to elute molecules from an ion exchange chromatography column?
pH or salt concentration
What is the disadvantage of reverse phase chromatography?
Not good for molecules with low solubility in water
Proteins can be separated based on charge using reverse phase chromatography.
False
Isoelectric focusing separates molecules based on size using a pH gradient.
False
Affinity chromatography is not suitable for all proteins.
True
Anion exchange resin has negatively charged groups on the beads, so it binds to cations.
False
Cation exchange resin moves slowly down the column when negatively charged molecules are added.
False
Ion exchange chromatography is generally used as a purification technique for lipids.
False
ELISA is a technique used to separate and quantify DNA molecules
False
Each well of the plate in indirect ELISA is coated with an antigen
True
Sandwich ELISA is used for quantitating the amount of antibody in a sample
False
The enzyme substrate turns yellow if the enzyme is not bound in sandwich ELISA
False
what does the pH needs to be for a molecule to bind to anion exchange resin?
greater than molecules isoelectric pH
what does the Ph need to be for a molecule to bind to cation exchange resin?
less than molecules isoelectric ph
what is the stationary phase in reverse phase chromatography?
hydrophobic beads
what is the mobile phase in reverse phase chromatography?
polar solvent
what does affinity chromatography beads have to attract the proteins of interest?
ligand attached to the beads
whats the first and secondary peak for in affinity chromatography?
first= non specific proteins second= when excess affinity ligand added
whats westeren blotting for?
find specific proteins within a mixture
proteins are already negatively charged due to SDS-PAGE in western blotting
True
Study Notes
-
Ion exchange chromatography: net charges of molecules change at different pH levels due to different pKa for COOH and NH2 groups.
-
To elute molecules from column, pH or salt concentration can be altered.
-
Isoelectric pH (pI): the pH at which a molecule has no net charge. Different amino acids have different pIs (e.g. aspartate at pH 3.5, lysine at pH 8.5).
-
Proteins separate based on charge using this method with a pH gradient to elute them one at a time.
-
Advantages: straightforward, good resolution, quick. Disadvantages: only works for charged molecules, some may not like pH or salt changes.
-
Isoelectric focusing: separates molecules based on charge using a pH gradient. Can be used with SDS-PAGE for size and charge separation.
-
Reverse phase chromatography: separates molecules based on polarity with hydrophobic beads and polar mobile phases. Advantages: flexibility, good resolution. Disadvantages: not good for molecules with low solubility in water.
-
TLC: thin layer chromatography separates molecules based on their interaction with a polar stationary phase and non-polar mobile phase. Advantages: can visualize components, different solvents can be used, cheap and easy. Disadvantages: small samples only, not good for proteins or large molecules.
-
Affinity chromatography: separates molecules based on their affinity for a specific ligand attached to stationary phase beads. Advantages: easy, quick, excellent purification. Disadvantages: not possible for all proteins.
-
Western blotting: visualizes specific proteins within a mixture using an antibody. Runs in conjunction with SDS-PAGE. Advantages: specific, sensitive. Disadvantages: not possible to extract protein, time-consuming.
-
Ion exchange chromatography: net charges of molecules change at different pH levels due to different pKa for COOH and NH2 groups.
-
To elute molecules from column, pH or salt concentration can be altered.
-
Isoelectric pH (pI): the pH at which a molecule has no net charge. Different amino acids have different pIs (e.g. aspartate at pH 3.5, lysine at pH 8.5).
-
Proteins separate based on charge using this method with a pH gradient to elute them one at a time.
-
Advantages: straightforward, good resolution, quick. Disadvantages: only works for charged molecules, some may not like pH or salt changes.
-
Isoelectric focusing: separates molecules based on charge using a pH gradient. Can be used with SDS-PAGE for size and charge separation.
-
Reverse phase chromatography: separates molecules based on polarity with hydrophobic beads and polar mobile phases. Advantages: flexibility, good resolution. Disadvantages: not good for molecules with low solubility in water.
-
TLC: thin layer chromatography separates molecules based on their interaction with a polar stationary phase and non-polar mobile phase. Advantages: can visualize components, different solvents can be used, cheap and easy. Disadvantages: small samples only, not good for proteins or large molecules.
-
Affinity chromatography: separates molecules based on their affinity for a specific ligand attached to stationary phase beads. Advantages: easy, quick, excellent purification. Disadvantages: not possible for all proteins.
-
Western blotting: visualizes specific proteins within a mixture using an antibody. Runs in conjunction with SDS-PAGE. Advantages: specific, sensitive. Disadvantages: not possible to extract protein, time-consuming.
Learn the fundamental principles of ion exchange chromatography, a technique used to separate molecules based on their charge or polarity. Explore the process involving a mobile phase running over a stationary phase composed of charged beads, allowing the separation of molecules according to affinity.
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