Chapter 5.2 Protein Purification and Analysis
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What properties may be used to measure (quanitfy) protein during purification in an assay?
What kinds of procedures take advantage of a protein's uniques structure and chemistry in order to separate it from other molecules?
What does increasing the salt concentration cause of proteins with different solubilities.
Proteins chemical or binding properties.
"Salting out" (precipitation)
To isolate a protein, you must first get it out of the cell and into solution. How can this be done? What two techniques are used to separate the molecules?
What is used when lipid is attached to the protein? What does it do?
- Disrupt the cell by crushing or grinding it.
- The protein is released but now have to separate by filtration or centrifugation.
A detergent, dissolve the lipid or solubilize it to recover the protein.
What are the three requirements for an assay?
- 1. Highly specific for target protein.
- 2. Highly sensitive.
- 3. Convenient to use.
What happens with a straightforward protein assay?
Are substances with colored or fluorescent products have been developed for this purpose?
If such a substance or product has no fluorescence available for the enzyme being assayed, what can be done and used to detect it? What is the reaction called?
The rate of the product formation is proportional to the amount of enzyme present, in other words, the enzymes catalyze reactions with readily detected products.
Use another enzyme to convert the product of the enzymatic reaction that has the fluorescence, coupled enzymatic reaction.
What are antibodies? What is the foreign substance that attaches to the antibody?
What are the atibodies recovered from?
When the protein binds to its corresponding antibodies, what kind of a mixture does the protein seem to be in?
Proteins produced by an animal's immune system in response to the introduction of a foreign substance, or antigen.
Blood serum of an immunized animal or from cultures of antibody-producing cells.
A complex mixture.
What kind of a technique is used when the protein, is indirectly detected by determining the degree to which it competes with a radioactively labeled standard for binding to the antibody.
What is the technique that uses an enzyme covalently attached to a second antibody?
Enzyme-linked immunosorbent assay.
What are the four steps to immunoassays?
On the fourth step, how do you assay the enzyme activity?
What is the detectible, substrate or product?
- 1. Immobilize the antibody to a solid surface.
- 2. Incubate with protein sample, to attach to antibody.
- 3. Add a second antibody that has a covalently linked assayable enzyme.
- 4. Wash and assay the enzyme activity.
The amount of substrate that is converted to product indicates the amount of protein present.
Using what equation can you determine the concentration of a substance in a solution and what is the type of spectroscopy?
What represents the optical density?
What is Io, epsilon?
What is plotted against what?
Beer-Lambert Law, Absorbance spectroscopy.
- Wavelength of incident light intensity.
- Absorptivity or extinction coefficient of solute at wavength
absorptivity (extinction coefficient, epsilon) or A vs. wavelength
What region do aromatic polypeptides absorb strongly in and what are the range of values? Why?
If e is known, what can be known?
If polypeptides do not absorb visible light (400-800 nm) what does that mean?
If there is a chromophore, what is the absorbance region that you can detect in and what does it mean for the protein detection?
Ultraviolet region at 200 to 400 nm. They have large extinction coefficient.
They are colorless.
In the visible region, used to detect proteins in mixture with other proteins.
Proteins are purified by what procedures?
What is the idea of protein purifications?
To eliminate selectively the other components of the mixture so that only the required substance remains.
What is the technique that is developed due to other substances showing absorbance of uv light, like nucleic acids?
What is the dye used and is the solution that the protein is in acidic or basic?
What wavelength does the coomassie brilliant blue cause the absorption shift appear in?
- Coomassie brilliant blue, acidic solution.
- 465-595 nm.
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