HI
My protein of interest is a secreted glycoprotein. So, amI correct in saying that it does not have a cellular localization. How is IHC done on sected proteins, how can they be detected by IHC? This may sound silly but I'm new to this concept.
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#2
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Sunflower
Posted 20 January 2010 - 05:32 PM
if I'm understanding you correctly, you're wanting to detect a protein after is has been secreted outside the cell and you're not looking for something membrane-bound?
are you using cells in suspension, like in culture, or are you staining tissue or adherent cells? if you're staining tissue cross-sections you may be able to perceive the protein as a halo around an individual cell. the only problem here is that when you permeabilize you might end up detecting membrane-bound protein in the process of being secreted, or even proto-protein inside the cell. if you want to use this technique I'm guessing you will have to find an antibody that only recognizes the mature, secreted form of the protein. I would also worry that fixing, washing, etc. would rinse the protein off the slide if it's not anchored in the membrane.
if you want to do suspension cells, I don't know what to tell you. I think WB of supernatant would be a better way to detect it.
if I'm wrong and you're looking for a membrane-bound protein, it's easy as pie. you'll see it as a halo.
are you using cells in suspension, like in culture, or are you staining tissue or adherent cells? if you're staining tissue cross-sections you may be able to perceive the protein as a halo around an individual cell. the only problem here is that when you permeabilize you might end up detecting membrane-bound protein in the process of being secreted, or even proto-protein inside the cell. if you want to use this technique I'm guessing you will have to find an antibody that only recognizes the mature, secreted form of the protein. I would also worry that fixing, washing, etc. would rinse the protein off the slide if it's not anchored in the membrane.
if you want to do suspension cells, I don't know what to tell you. I think WB of supernatant would be a better way to detect it.
if I'm wrong and you're looking for a membrane-bound protein, it's easy as pie. you'll see it as a halo.
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#3
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Posted 21 January 2010 - 05:43 PM
aimikins, on Jan 21 2010, 09:32 AM, said:
if I'm understanding you correctly, you're wanting to detect a protein after is has been secreted outside the cell and you're not looking for something membrane-bound?
are you using cells in suspension, like in culture, or are you staining tissue or adherent cells? if you're staining tissue cross-sections you may be able to perceive the protein as a halo around an individual cell. the only problem here is that when you permeabilize you might end up detecting membrane-bound protein in the process of being secreted, or even proto-protein inside the cell. if you want to use this technique I'm guessing you will have to find an antibody that only recognizes the mature, secreted form of the protein. I would also worry that fixing, washing, etc. would rinse the protein off the slide if it's not anchored in the membrane.
if you want to do suspension cells, I don't know what to tell you. I think WB of supernatant would be a better way to detect it.
if I'm wrong and you're looking for a membrane-bound protein, it's easy as pie. you'll see it as a halo.
are you using cells in suspension, like in culture, or are you staining tissue or adherent cells? if you're staining tissue cross-sections you may be able to perceive the protein as a halo around an individual cell. the only problem here is that when you permeabilize you might end up detecting membrane-bound protein in the process of being secreted, or even proto-protein inside the cell. if you want to use this technique I'm guessing you will have to find an antibody that only recognizes the mature, secreted form of the protein. I would also worry that fixing, washing, etc. would rinse the protein off the slide if it's not anchored in the membrane.
if you want to do suspension cells, I don't know what to tell you. I think WB of supernatant would be a better way to detect it.
if I'm wrong and you're looking for a membrane-bound protein, it's easy as pie. you'll see it as a halo.
Thanks for your response. My gene of interest has reduced mRNA expression in cancer tissue compared to non-tumor tissues and this gene codes for a secreted protein. I want to use IHC staining to check if there is any correlation between my protein and beta catenin expression in tissues. I'm new to this and I'm not sure if a secreted protein can be detected by IHC.
#4
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Posted 22 January 2010 - 09:10 AM
If the protein is being actively produced, you should be able to see some in the cytoplasm that hasn't been secreted. If that doesn't work, your best bet would be Western or ELISA or culture supernatant.
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