State the main conclusion from the figure and state how it relates to Huntington
ID: 201760 • Letter: S
Question
State the main conclusion from the figure and state how it relates to Huntington’s Disease. Please answer in detail as I am confused on how to read western blots. Huntington Figure 5 from Tissue-Specific Proteolysis of Huntingtin (htt) in Human Brain: Evidence of Enhanced Levels ofN- and C-Terminal htt Fragments in Huntington's Disease Striatum The Journal of Neuroscience, 15 March 2001, 21(6): 1830-1837; http://www.jneurosci.org/content/21/6/1830.long Fig. 5. 3 250 Striatum (putamen): huntingtin fragments detected with antibodies recognizing the N terminus, N- terminal domain, and C-terminal domain of htt. Striatum (putamen) tissue homogenates from control (lanes 1, 3, 5) and HD (lanes 2, 4 6) brains were analyzed by Western biots with anti-(1-17) serum (lanes 1, 2), N- terminal domain 210 200 kDa 150120-116 100 -66 -55 -36 50 45 -50 45 35 20 -21 Tissue: C HD C HD N-domain C HD C-domain Antibody: (1-17) View larger version: In a new window Download as PowerPoint Slide antibody (lanes 3, 4), and C-terminal domain antibody (lanes 5, 6). Each laneof the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (18 ug).
Explanation / Answer
The use of western blots for identification and quantitation of Htt protein has been used successfully. Type of gels that is used for separation, the antibodies used, and specific protocol modification sized according to the type of cell line and length of mutant Htt protein
The Htt protein is tagged with myc at its C-terminal end. The cells are maintained in a culture medium that contains doxycycline, and mutant protein production is induced when doxycycline has been removed from the culture medium.
The identification and quantitation of mutant Htt protein by western blotting both in vitro in COS-7 cells and in vivo from the striatum of mice brain following treatment with siRNA. Following the standard process cell lysis procedures, the lysates are loaded onto gradient 5–10% SDS-polyacrylamide gels and transferred to Immobilon-P membranes after resolution. The membranes were incubated with primary antibody against the Htt protein (MAB 5374) and visualized in a chemiimager. The western blotting approach was successful in quantifying the levels of mutant Htt protein in the control as well as siRNA treatment groups
The detection process and the quantitation of Htt protein with western blotting have been proved to be effective.The choice of the antibody varies based on the nature of the mutant Htt produced and the presence of reporter genes. The western blotting principles are well established, and care should be taken to develop a robust protocol that would yield reproducible results.
Reading the Western Blot Results
On the bsis of infection or illness being tested for, there may be several different bands reported from the Western blot, each with either a positive or a negative result. There are some bands reported for the Western blot which are not indicative of a result. Ensure the information provided by the clinician informs which blots are key.
Look for the sizes of the bands. They will be represented by number, which is followed by "kDa" or preceded by "p." This is the size of the protein which is been detected and is the scale on which the proteins are separated in a Western blot. It is these differently numbered bands that represent different proteins and will determine a positive result or not.
Determine which bands have a positive result and what this means to us.say for example,test for Lyme disease, we can see several bands different from each other which may produce a positive result, and any one of these will mean a positive result. Other tests are simply produce non-significant bands. These may not be reported, but ensure it is clear which are the important bands. Specific bands which show a positive result means a positive result for the infection that was being tested for.
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