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Post subject: Protein Detection/Separation Methods
Dear all,
Does anyone know the exact comparison between Western blot, immunofluorescent, immunohistochemistry, and ELISA in terms of their application and when/why should use this method instead of others? Well, their basic application is to detect the presence of specific proteins in a biological sample (tissue, cell lines, or individual cells).
I have a doubt on how to apply these methods. Let say, I want to check the presence of cytokines (protein) expressed by pancreatic cancer cells, then I perform Western blot to check their presence (intensity of the bands with marker/ladder). Could I replace Western blot analysis with immunofluorescent staining or immunohistochemistry since they share the same basic purpose?
Please share yours knowledge! Thank you!
Sat Jul 30, 2011 5:56 pm
gula
Rank: Member
Joined: Mar 20, 2008
Posts: 94
Post subject:
Immunocytochemistry and immunhistochemistry show both the detection of antigens in situ. Histo = for example tissue-section, cyto = isolated cells or cells collected with brushes, in urin, in cysts...
IHC is mainly a qualitative assay. Some antigens can be determined in a semiquantitative manner (+,++) but not in figures.
For blot-techniques you have to extract your antigen of interest out of the tissue. Therefore you loose the morphology. To be sure, that the antigen is only tested in the cells of interest, laser capturing of the cells is necessary eventually.
For ELISA-testing (as far as I remember) the antibody is bound on a tube-surface. The antigen has to be extracted - so again no morpholgy-context. With comparison to a known concentration and standard curve, you can determine the quantitiy of your antigen.
I think an important point is, if your cytokines are bound well enough in the tissue, so any IHC-procedure would not result in antigen-loss. Or if a suitabled fixation will hold the antigens in position.
And if morphology is important to your test-interpretation, or if quantification is the main thing.
gula
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