Please answer C-E in detail. Thanks You are interested in studving the expressio

Question

Please answer C-E in detail. Thanks

You are interested in studving the expression of the Abcal gene in mice at the RNA level. In order to study Abcal you isolate total RNA from the following tissues and perform a Northerm blot. You probe first for Abcal. After you have results from that experiment (upper box), you strip the probe off the membrane and reprobe for GAPDH. Those results are shown in the lower 1 2 3 45 6 7 7000 nt Abca1 Gandh | 400 nt Figure 1. Expression Analysis of Mouse Abc1 by Northern Blotting. Lanes 1-7 represent total RNA isolated from the following specific tissues in the adult mouse: 1)Liver 2)Spleen 3)Large Intestine 4)Lung 5)Heart 6)Brain 7)Thymus.. A. GAPDH stands for Glyceraldehyde 3-phosphate dehydrogenase, which is an enzyme expressed at a high level in all cells (it is needed for general cell maintenance). Why did you do a subsequent probing for GADPH? B.Which tissues express Abcal? Which has the highest expression? What is shown on the gel that allows you to conclude this? C. Describe in general terms) the probe used in the Northern blot for Abcal. D. What tissues do not contain Abac1? Explain what leads you to that conclusion E. You want to determine the subcellular localization of the ABCA1 protein in cells of the thymus using antibodies, briefly how would you do this?

Explanation / Answer

Northern blot Probe is a technique to study gene expression via RNA.

RNA is extracted from the cells and ran out trough an electrophoresis gel that contains formaldehyde to ensure linear conformation. While the RNA is in the gel, it is exposed to a RNA probe that contains a specific sequence and is radioactively labeled. This is to identify the specific RNA sequences in the sample. In this case, the ones for ABCA1 sequence.

After electrophoresis process, a nitrocellulose membrane is placed at the top of the gel. The positive charge of the membrane attracts RNA which is negatively-charged and bind together. UV light is used to ensure this linkage.

Paper towels and weight are placed on top as well. This is because there is generated a high water potential and a low water potential lets the Buffer to transfer the RNA from the gel to the membrane.

Within the membrane, the radioactive probe binds to the sample only if it contain the specific sequence that was probed for.

Then the membrane is washed to avoid any background noise.

The hybrid signals are detected by X-ray film and quantified by densitometry.

The Immunostaining is the method used to detect protein expression in different tissues or organelles. It is based in the principle of antibodies carrying a fluorescent dye binding to specific proteins. When the labeled antibodies are added to an histological sample, they bind to the specific proteins that were designed to staining the cells and the organelles that contains such protein making visible its presence in the sample.

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