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31 Cards in this Set
- Front
- Back
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What 3 properties should an Assay have?
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1) Specific - targets only your protein
2) Sensitive - low amt. of protein needed. 3) Easy to do - process will likely need to be repeated. |
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What are the 4 main types of Assays?
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1) Binding assay
2) Enzyme assay 3) Immunological assay 4) Biological assay |
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What was the example of a Biological Assay we talked about?
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Testing for presence of NGF (Neural Growth Factor).
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What was the example of an Immunological Assay we talked about?
How does it work? (4 steps) |
ELISA - Enzyme-Linked Immunosorbent Assay
(pg. 95) 1) An antibody against the protein of interest is immobilized on an inert solid such as polystyrene. 2) The solution to be assayed is applied to the antibody coated surface. The antibody binds the protein of interest, and other proteins are washed away. 3) The protein-antibody complex is reacted with a second protein specific antibody to which an enzyme is attached. 4) Binding of the second antibody-enzyme complex is measured by assaying the activity of the enzyme. The amount of substrate converted to product indicates the amount of protein present. |
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What are the 5 steps of protein purification?
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1) Starting material
2) Develop assay 3) Extraction 4) Try separation techniques 5) Bookkeeping |
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What are 3 examples of starting material we talked about?
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1) Recombinant source (cloned genes)
2) Animal tissue (unknown protein) 3) Tissue culture cells (unknown protein) |
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What 3 results do you want in your bookkeeping section?
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1) Specific activity
(total activity) / (amount of protein) 2) Yield (activity after) / (activity before) 3) Fold purification (specific activity after) / (specific activity before) |
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What are 5 protein characteristics are considered when looking for a separation technique?
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1) Solubility
2) Ionic charge 3) Polarity 4) Size 5) Binding specificity |
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Separation techniques: Name the purification procedure(s) talked about for:
Solubility |
Salting out
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Separation techniques: Name the purification procedure(s) talked about for:
Ionic charge |
- Ion exchange chromotography
- Electrophoresis - Isoelectric focusing |
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Separation techniques: Name the purification procedure(s) talked about for:
Polarity |
Hydrophobic interaction chromatography
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Separation techniques: Name the purification procedure(s) talked about for:
Size |
Gel Filtration Chromatography
SDS - Page Ultracentrifugation |
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Separation techniques: Name the purification procedure(s) talked about for:
Binding specificity |
Affinity Chromatography
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Salting out |
Protein characteristic: Solubility
EXA from pg. 97 (a) (NH₄)₂SO₄ is added to a solution of macromolecules to a concentration just below the precipitation point of the protein of interest. Centrifuge. (b) After centrifugation, the unwanted precipitated proteins (red spheres) are discarded and more salt is added to the supernatant to a concentration sufficient to salt out the desired protein (gray spheres) (c) After a second centrifugation, the protein is recovered as a precipitate, and the supernatant is discarded. |
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Separation techniques: Purification procedures
Name the 4 types of column chromatography talked about, and which protein characteristic they filter based on. |
1) Ion exchange chromatography
(Ionic charge) 2) Hydrophobic interaction chromatography (Polarity/hydrophobicity) 3) Gel-filtration chromatography (size) 4) Affinity chromatography (binding specificity/specific interaction) |
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Ion-exchange chromatography |
Protein characteristic: Ionic charge
- Separates anions and cations - Charged molecules bind to opposite charged groups on the column - Affinity based on presence of salt and pH. pg. 99 (a) A mixture of proteins dissolved in a small volume of buffer is applied to the top of the matrix in the column (b) As elution progresses, the proteins separate into discrete bands as a result of their different affinities for the exchanger. (c) Salt concentration in the elutant is increased to elute the remaining proteins. * All proteins are separated into different vials based on their affinity for the salt (exchanger) |
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Define:
Elute |
To remove by dissolving.
To extract (one material) from another, usually by means of a solvent |
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Gel filtration chromatography |
Protein characteristic: Size
- Separates molecules according to size. pg. 100 (a) Gel beads with tiny holes are in a tube. Small stuff fits in (your protein), big stuff doesn't. (b) Sample solution is applied to top of column (c) The small molecules can penetrate the gel and consequently migrate through the column more slowly than the large molecules. (d) Large molecules are eluted first and collected as fractions. Small molecules require a larger volume of solvent to elute.` |
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Affinity chromatography |
Protein characteristic: Binding specificity
- Exploits specific binding behavior. pg. 101 - Column contains a specific ligand for protein of interest. Ligand is attached to a matrix. - Impure mixture of proteins runs through the column - Proteins with affinity for the ligand stay behind. - To free protein from ligand, wash with an elution mixture of one of following: (a) solution of high conc. of free ligand (b) change of pH (c) salt concentration |
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Define:
Ligand |
A molecule that binds to a receptor
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Ultracentrifugation |
Protein characteristic: Size
- Spin vial real fast; 100k rpm - Sedimentation rate varies with mass and shape of protein. Also varies with density of medium. |
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Define:
Electrophoresis |
The migration of ions in an electric field.
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
Gel Electrophoresis (PAGE) |
Protein characteristic: Size
pg. 101-102 for more details - Gel acts as a mesh or sponge - Proteins are driven through gel by an electric field - Proteins are detected by staining, autoradiogrpahy, or immunoblotting |
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Separation techniques: Describe the purification procedure below. Name the protein characteristic it's a procedure for.
SDS-PAGE |
Protein characteristic: Size
pg. 102 for more details - Proteins are denatured by boiling in SDS - Charge/Mass ratios are the same, which allows for filtration based on molecular mass. |
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Endgroup analysis:
What is one method that can be used to determine the AA of the N-terminus on a protein? |
Dansyl chloride reaction
pg. 106 |
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Endgroup analysis:
Describe the Edman degradation method |
pg. 109
- A way to determine amino acid sequence of a protein once it's isolated (a) PITC reacts with the N-terminal amino group to form PTC. i.e. PITC sticks to polypeptide and forms PTC (b1) Now acid is used on PTC to cleave N-terminus. (b2) Doesn't hydrolyze the rest of the AA, thus you just lost the n-terminus. (c) Thiazolinone-AA complex is converted to PTH (d) AA identified through chromatography. i.e. Identifying PTH will identify the amino acid attached within it. - Thus with repeated steps of Edman degradation, a ppt. sequence can be determined. |
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Describe 4 characteristics of protein evolution
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- Random mutations result in changes in protein sequence
- Reduced fitness leads to loss of that mutant strain - Increased fitness leads to increased frequency of that strain - Proteins slowly change during evolution |
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What is the lower limit for protein residues? The upper limit? Why?
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100 - 1000 residues
40 residues is around the lowest number a protein can have and still be stable. The large the residue number, the less efficient it is to make the protein, and the change of errors increases too. |
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Define:
Adsorption |
The accumulation of gases, liquids or solutes to the surface of a solid or liquid.
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What are 5 factors to account for when attempting to purify a protein? Briefly say why for each one.
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(1) pH
Could denature protein. (2) Temperature Could denature protein. Some proteins denature at only a few degrees higher than their antive environment. (3) Presence of degrative enzymes Proteases and nucleases. Can be controlled with temperature or pH to inactivate them, or by adding compounds that block them. (4) Adsorption to surfaces Many proteins are denatured by air/water interface, or with glass/plastic surfaces. (5) Long term storage Slow oxidation and microbial contamination must be prevented. |
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Define:
Primary Protein Structure |
The sequence of amino acids within a polypeptide chain.
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