A curious result- hide and seek with my protein - (Oct/06/2008 )
Hi,
I have a strange phenomenon. I wanted to determine the expression of a protein by immunohistochemistry (ihc) and immunoblot. For both analyiss I used the same primary antibody. The result is that I could detect immunohistochemically my protein on cultured tumor cells but I failed in the immunoblot. What is the reason for that strange thing. I am really helpless. Tiffy
Is your antibody qualified for IB? Some Ab only work well in IHC or IB, some do in both. The company normally gives advice to this. Have you done IB before with this Ab? Try to establish it first with a positive control which gives you a good, strong signal, then go on with your sample. Try different condition for Ab-concentration, duration of incubation etc. if it will not work well directly.
The antibody works for both and moreover it worked well on an other cell line. I wonder which result is wrong. Or how can i check it. Maybe with third method such as Q-PCR?
How often have you done the IB? Sometimes cell lines differ a lot and you have to reestablish the Ab for the new one. IB can be difficult, do not give up to early. But sure, you can do other experiments to confirm your results on RNA level. Is the IHC reproducible? Then just use this method for protein detection if the IB will not work at all.
Yes, i got reproducible results using the ihc. I carried out the ib three times (each run with two flasks). Could it be that the denaturation state causes the result? I mean i don´t denature the cells for the ihc but the protein samples. Does it make sense to run a sds-page under non-denaturating conditions? Tiffy
that would be native-page. you can try it, but, you said that you were able to detect the protein from other cell lines with immunoblot, wasn't that sds-page?
my first thought, when i read your initial post, was that the antibody was directed toward an epitope that is not available when the protein is denatured. in that case you would need a different antibody. native-page may answer the question.
that would be native-page. you can try it, but, you said that you were able to detect the protein from other cell lines with immunoblot, wasn't that sds-page?
my first thought, when i read your initial post, was that the antibody was directed toward an epitope that is not available when the protein is denatured. in that case you would need a different antibody. native-page may answer the question.
Yes, I could detect the protein in other cell line by sds-page. Is it possible that a protein can differ strongly in that way?
is your antibody polyclonal, monoclonal or mixed monoclonals?
are your cell cultures from different species?
denaturing proteins with sds can distort a 3 dimensional epitope, making the binding of an antibody less certain.
are your cell cultures from different species?
denaturing proteins with sds can distort a 3 dimensional epitope, making the binding of an antibody less certain.
It could also due to the amount of protein in your interested cell line is not enough for it to be detected by IB. Another possibility is that in your interested cell line, your protein may be modified (different states of phosphorylation, for example) somehow differently from the other cell line (the one that you could detect by IB). The modification may be changed during the process of lysis and IB but not IF and critically affect the epitope. May need to try different conditions for IB: lysis buffer, blocking...
Hello,
I have got new infos about my problem. I ran a simple experimet to determine whether the denaturation of my protein causes the loss of my IB signal. I have harvested both cell lines in non-denaturing lysis buffer. Then I dropped an equal amount of protein (100µg) of a nitrocellulose membrane, dry.(No PAGE, no blotting) Subsequently I carried out the normal procedure, blocking, 1 ab wash 2 ab. The result was that the one cell line has a good signal the other one not.
What would be the next step?
Tiffy