University of North Texas Protein Identification Techniques Worksheet

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Homework Protein Identification Techniques (Chapter 5)- 30 points total A minimum of three pages required, and you must answer all questions to receive 30 points. Please provide the reference on the fourth page. Students who submitted the assignment after deadline will get 2 points deduction each day after the deadline date. The deadline for this assignment is June 23 by 11:59pm. 1. What is the basis for the technique called ELISA(Enzyme-Linked Immunosorbent Assay)? – 5 points 2. What is the difference between a primary antibody and a secondary antibody? – 5 points 3. What are some ways that the antibody-protein complex can be visualized in ELISA or western blots? – 5 points 4. What are the main procedures involved in a western blot? – 5 points 5. ______________________, or ________________ take the same concepts we have seen with the ELISA and western blot and add the power of tremendous throughput. A typical ELISA experiment uses a 96 well plate, so each experiment can look at 100 samples. _______________, on the other hand. May have 30,000 separate samples stuck on a chip a few centimeters on a side. Fluorescence is the most common way to see the results. – 3 points 6. __________________ is a technique using antibodies. In this case proteins are separated by gel electrophoresis and then the protein bands transferred to a thin membrane. The membrane is reacted with a specific antibody to identify where on the original gel the protein of interest was located. The ____________blotting technique was developed for the transfer of DNA from a gel onto nitrocellulose. The next blot to be designed transferred RNA, it was called ______________. – 3 points 7. What is proteomics? – 3 points Provide the references you used (1 point)
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Homework: Protein Identification Techniques

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Question One
What is the basis for the technique called ELISA(Enzyme-Linked Immunosorbent Assay)? – 5 points
ELISA (Enzyme Immunoassay) is an assay that analysts use measure and detect certain proteins,
peptides, hormones, and antibodies in the blood (Paulie & Perlmann, 2016). ELISA yields
quantitatively and distinguish specific and non-specific interactions by deploying solid surfaces
to serial binding. While deploying ELISA, it is believed that specific antibodies will bind a specific
antigen. Therefore, whenever such an antibody is deployed (and the target antigen is present)
they will bind, thus showing that it is available. It is worth mentioning that one can also
understand the quantity of such antibodies by determining the amount of antigen that bonded.
To fully understand ELISA, its three types have been discussed below.
There exist three basic types of ELISA namely, indirect ELISA, Sandwich ELISA, and Competitive
ELISA (Paulie & Perlmann, 2016). Indirect ELISA is commonly deployed to detect whether a
sample is characterized by an antibody. In this case, a sample to be tested is placed into a
microtiter plate whose wells have been coated with an antigen. Once the primary antibody has
been washed away, an antigen-antibody complex is detected after a substrate that gives a
specific color is added. A Sandwich ELISA is commonly deployed to determine whether a sample
is characterized by an antigen. In this case, a well whose wells are coated with an antibody are
deployed. One the sample has been passed through the well, an enzyme-specific substrate is
added to give a specific color to the deposits. Finally, a Competitive ELISA is deployed to
determine the concentration of an antigen is in a sample. The difference between a Sandwich
ELISA and a Competitive ELISA is that the enzyme-linked antigen determines the number of
primary antibodies within the well.


Question Two
What is the difference between a primary antibody and a secondary antibody? – 5 points
The main difference between primary and secondary antibodies is that a secondary antibody
binds with the primary antibody whereas a primary antibody binds to a specific antigen (Levoir
& Goo, 2020). The difference is as a result of the procedures each antibody follows to
production. The procedures have been discussed below.
Whenever one wishes to produce a primary antibody, they are required to immunize a host
species (like a chicken, goat, mouse, or a rabbit) against an antigen (Levoir & Goo, 2020). For
the secondary antibodies, the production is totally different because the antibody is injected
directly from one host species to the other. For instance, whenever the antibody was generated
inside a chicken, the secondary antibody would be generated in a mouse. Another issue that
could be used to determi...

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