Identification Terms and Essays Chapter One
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Antibodies that are produced by different cells responding to the same antigen at different epitopes on the antigen
Useful for detecting antigens that are scarce or in low abundance
For more specificity, these are made against antigens
First step in an isolation that disprups cells in a homogenizer and results in a mixture called the homogenate that undergoes centrifugation to result in a pellet
A test that is performed to detect our sample in solution, as well as the specific activity
The ratio of enzyme activity to the amount of protein in a mixture; determined by dividing total activity by total protein; it should increase with purification. Of the protein I have, how much is mine (units/ mg)
If we know that our protein of interest is capable of binding to a specific molecule or sugar, for example, glucose, we can add beads containing this specific tag. Therefore, the protein will bind; we will run a buffer through it to wash the sample of unbound; and, to detach, we will add a glucose solution, causing the proteins to detach.
In column chromatography, it is put in the column to basically cleanse and lubricate. Generally, it helps to wash out any left-over proteins from a previous experiment. It can also help to separate the fractions that are collected; specific to the type of test
Sodium dodecyl sulfate; this is used in gel electrophoresis. The mixtureof proteins are first dissolved in a solution of SDS, which is a detergent that disrupts nearly all noncovalent interactions in native proteins. Eventually, after beta mercaptoethanol dissolves any disulfide bridges present, SDS binds to the denatured proteins at a ratio of 1 SDS anion for every two amino acid residues. This creates a negative charge on the molecule, which will migrate towards the positive end of the gel
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions
1) Why are monoclonal antibodies so difficult to isolate? What is a way to bypass this problem?
- a. Monoclonal antibodies are so difficult to isolate because they do not live long. So, to produce a clone of cells producing one specific antibody, it is not possible since they do not live long. A way to overcome this problem, however, is by fusing the cell that is producing the specific antibody to a cancerous cell that is generally immortal and will continue to divide. This new cell is called a hybridoma; and, it can be used to produce clones of the single cell that is specifically producing that antibody
- b. Inject antigen into animal (mouse, for example); remove spleen and extract plasma cells and fuse them to myeloma cells, creating hybridoma cells, which indefinitely produces the indentical antibody specified by the parent cell from the spleen. Because this creates a collection of cells that produce the desired antibody, we subdivide and reassay until a pure cell line develops.
1) Which is the strongest chromatography technique?
1) What does recombinant technology enable?
- a. it enables proteins to be expressed in large quantities (by the manipulation of the primary sequence to produce proteins that weren’t originally produced)
- b. affinity tags can be fused to proteins
- c. proteins with modified primary structures can be readily generated
1) Why is knowing the amino acid sequence valuable?
- a. The sequence of a protein of interest canbe compared with all other known sequences to ascertain whether significant similarities exist
- b. Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathways
- c. Amino acid sequences can be searched for the presence of internal repeats
- d. Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing
- e. Sequence data provide a basis for preparing antibodies specific for a protein of interest
- f. Amino acid sequences are valuable for making DNA probes that are specific for the gene encoding the corresponding proteins
1) What are the basic steps of characterization?
- a. Get the amino acid composition
- i. Hydrolyze the amino acid bonds
- ii. Attach indicator dye to see color and allow spec to read it
- iii. Run through an HPLC with a spectrophotometer at the end, which will read the amino acids as they exit the HPLC.
- 1. The time they come out and their elution will determine the type of amino acid it is
1) Why is it useful to use several isolation techniques?
The reason for this is because one step is not sufficient. Several proteins in the crude mixture will likely co-elute with the desired material. Therefore, to purify the sample, we must take advantage of its specific properties that will allow it to be isolated from other proteins in the solution. For example, because gel-filtration may result in numerous proteins with various sizes, we can use ion exchange chromatography if our protein has a charge, or even affinity chromatography if our sample is very specific for what it binds.
1) What is special about HPLC?
- a. You can separate molecules differently with greater differences or slight differences
- b. Much better resolution and able to use much smaller quantities for isolation
1) How is the gel made? Why is it used?
They are used because they are chemically inert and readily formed by polymerization of acrylamide with a small amount of the cross-linking agent methylenebisacrylamide to form a 3D mesh
When assaying our mixture, what do we want to ask and make sure of?
- a. We want to make sure that, as the total protein goes down, our sample is still located in the total protein. Also, we want to ask how much of the specific activity corresponds to my protein. This should go up as the purification proceeds. Essentially, we want to make sure that our total protein goes down, but our total activity stays as high as possible; and, we want our specific activity to increase
- b. We want to make sure that, at each step, when we run the gel, we see less of the total bands, but more of our bands
Explain the Edman degradation in detail.
The Edman degradation sequentially removes one resi- due at a time from the amino end of a peptide. Phenyl isothio- cyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative. Then, under mildly acidic condi- tions, a cyclic derivative of the terminal amino acid is liberated, which leaves an intact peptide shortened by one amino acid. The cyclic compound is a phenylthiohydantoin (PTH)–amino acid, which can be identified by chro- matographic methods. The Edman procedure can then be repeated on the shortened peptide, yielding another PTH–amino acid, which can again be identified by chromatography. They are passed through HPLC, which reads PTH-amino acids rapidly
Why does knowing the DNA sequence not really help when it comes to proteins?
The reason is because DNA sequences do not account for the many modifications of proteins. It just includes the nascent protein without modification. Therefore, more chemical analyses must be done to determine the native conformation
Explain how monoclonal antibodies has opened new vistas in biology and medicine?
- a. The hybridoma method of producing monoclonal antibodies has enabled large amounts of identical antibodies with specificities to be prepared. They provide insight into relations between antibody structure and specificity. They also can serve as precise analytical and preparative reagents.
- b. For example, proteins that guide development have been identified with the use of monoclonal antibodies as tags. They attach to solid supports and could be used as affinity columns to purify scarce proteins.
- c. In a fruit fly embryo, the embryo was stained with an antibody for the DNA-binding protein encoded by engrailed, an essential gene that specifies body plan.
- d. This was done by taking a drop of buffer and making a puddle on top of the unstained section. The molecule binds an antibody, which can’t be seen until a secondary antibody with bound substrate is attached. Precipitates then form and create band where the protein’s location was.
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