1) You have used primary antibodies raised in mouse (for GAPDH, 35.8 kda) and in
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Question
1) You have used primary antibodies raised in mouse (for GAPDH, 35.8 kda) and in rabbit (for Cyt P450 reductase, 77 kDa) followed by a treatment with 2 types secondary antibodies each specific for 1 primary antibody and each labeled with a different infrared-fluorescent molecule. Would you still be able to distinguish GADH from Cyt P450 reductase, if the two primary antibodies were both raised in mouse and as a consequence a single type of secondary (anti-mouse) was used? Explain.
2) Suggest two experiments confirming that the Cyt P450 reductase you detected is associated with mitochondria. You don’t have to give detailed experimental protocols, but you need to indicate controls that needs to be included in your experiment.
3) Could the measure of ATP production be one of these experiments? Explain
Explanation / Answer
If both the antibodies are raised in mouse we can still detect the GAPDH and CytP450, The primary antibodies are bind specifically with their respective targets at 35.8 kda and 77 kda respectively. These two bands are wide enough to separated from each other in a SDS-PAGE. When the membrane is incubated with anti-mouse secondary antibody both the primary antibody are bound with the secondary and they can be clearly observed under infrared ray. Association of CytP450 can be proved with the help of two under mentioned methods: a)Immunohistochemistry (IHC): In this technique the primary antibodies are allowed to bind with the target protein in the cells and then with the help of a fluropore tagged secondary antibody the position of the target protein is detected under florescence or confocal microscope. Stain the cell with an antibody against a known mitochondrial protein (Like COX41, Cytochrome C Oxidase, HIF-1 alpha etc.) as well as with an antibody against CytP450 and after detecting the position of the protein in the cell overlay the images. If the images are overlapped then it’s proved that CytP450 is present with mitochondria. b)Cell Fraction technique: Using a cell fractionation we can separate the cellular organelle. Collect the all fraction separately and run all of the fraction in a SDS-PAGE and detect them using a primary antibody against CytP450. Only the mitochondrial fraction shows the positive result. Production of ATP can be also used as another technique. From the fractionated samples only the mitochondrial fraction shows the positive in case of ATP production as well as presence of CytP450 protein.
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