biology question #3025



Rafael Sumalinog, a 16 year old male from Edmonton asks on November 6, 2005,

Q:

What is immunofluorescence analysis, what basic materials are used, and how long does it take to perform? Is this similar with cytometric assay and immunoblot analysis in terms of determining how a chemical affects a cell (eg. disabling motility and morphology)? Also, how long would cytometric assay and immunoblot analysis take, and what are they for? And finally, do you know if these (or similar) assays can be done in Edmonton?

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the answer

Wendy Hutchins answered on November 7, 2005, A:

Immunofluorescence is very similar to immunoblot - but the big change is that we usually do it on fixed tissue or cells rather than proteins that were blotted onto a piece of paper. Cytometric implies "movement" to me, thus this term likely encompases functional assays on cells when they are alive, unless you are talking about flow cytometry - in which again, the way we make things visible is very very similar and can be done on either live or fixed cells. Flow cytometry uses a very expensive instrument for detection and works on single cells.

Let's see why...

First to understand is that the immuno part of any of these assays is the product of an immune response to something foreign. To use these products, antibodies, we would have to have the protein or other molecule in pure form, then we would inject it into an animal from another spieces. For example, we want to test beta-actin in human cells, so we would inject it into a mouse, or rat, or goat. Let's say mouse. The mouse will make an anti-human beta actin antibody. We can purify that from the mouse's blood (whole companies exist that you can buy these from!). Another company will take mouse antibodies and inject them into a goat. The goat will make an anti-mouse antibody that will bind the mouse antibody. Now, for us to see the antibody made in the goat, we will attach a tag to it. Tags can be fluorescent, an enzyme, or even a bead.

Now to do the test...

Take human cells and fix them with various agents such as methanol or formaldeheyde (10 min). Wash. Put some mouse or goat sera on the cells to block proteins sticking where they are not supposed to (30 min-1 hour). Wash. Put on the anti-beta actin antibody we made in the mouse (30-60min). Wash. Put on the anti-mouse antibody we made in the goat that has the tag on it (30-60 min). Wash. If this were a piece of tissue on a slide, we would look at it using a fluorescent microscope. If we were doing flow cytometry, we would take it to the machine and have someone run it for us. If this were an immunoblot, we would use a stain to see it.

Here is a picture of how immunofluorescence works and one on Western blotting a form of immunoblot.

All of these assays are available in Edmonton probably at the University of Alberta. If you are thinking school project, I would not go much further with flow cytometry as it is very expensive for most applications and likely would not be offered to you. The other two assays are common and will only be as expensive as that first mouse antibody. Some of these are not cheap, but 1ML will be reasonable (like $200) and do thousands of assays as compared to the same price for 1 flow cytometry test. Perhaps you should find someone who is studying your protein of interest and see if they have some antibody already! I would check out the webpages of the faculty at UofA to find the right people. Alternately, if you don't find them easily, contact someone at University of Alberta Outreach.

 

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