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Immunofluorescence microscopy, Western Blot, ELISA, ... - (Jul/07/2006 )

Would you please explain for me the differences in the application of these methods: Immunofluorescence microscopy, Western Blot, ELISA ?


With immuno fluroescence microscopy, one can detect the antigen in cells.

With Western Blot, one detects antigen from total protein extracts

With ELISA, one can detect antigen from a given sample like serum


Can't we use ELISA instead of Western blot and vice versa?


well strictly although the techniques all measure the same things eg a certain protien, the way the data is presented varies, e.g. ELISA gives you quantifiable data (actual numbers), while western blot readings are less quantifiable as it is more a a visual assay

so what process you use depends on what type of results you are looking for

-grapes of wrath-

ELISA : capture a specific protein with antibodies adsorbed on the surface of a well. Detect the protein with antibodies coupled to an enzyme. Add the substrate of the enzyme.
the result is a color proportionnal to the quantity of protein you have captured.
With ELISA you can quantify, but you do not see how much the antibody captured specifically your protein.

Western-blot : you separate your proteins on a gel, depending on their molecular weight, and then you detect your protein with an antibody and a second antibody coupled to an enzyme. It's less sensitive than ELISA, you can semi quantify, but not as good as ELISA, however, you see that your detection is specific because you see only one band, at the right molecular weight.

immunofluorescence microscopy : used to localize the protein inside the cell.