i am aware that IF may not be that quantitative to compare expression levels but merely for detection (present or absent) is IF more sensitive than western blot?
im habdling a cell line that express a particular protein. i have tried detecting by western blot this protein but coulnt not get any signal. whereas for IF, i could see nuclear staining, although weak. The inventors of this cell line also used IF to show that this protein is expressed, omitting (strangely) any western blot analysis (i guess its either they didnt bother to do OR similarly they couldnt detect it)
Also remember that western blotting cannot detect conformational epitopes ...
Also remember that western blotting cannot detect conformational epitopes ...
Hi Iwan,
It happened to me a lot of times not getting WB signal but seeing positive IF. As in IF you can spot cellular amounts of protein it is indeed more sensitive than WB.
If you want to keep trying see it on WB you could perform rupture with silicon carbide followed by diferential centrifugation. By this you'll be able to get an enriched and concentrated nuclear fraction that may improve the detection by WB.
About the dim signal in the IF, it would be great to perform a titration of the primary antibody in advance to achieve a better signal. Using the antibody less diluted than the curent use and testing a couple more of dilutions should be enough to find an optim titre. Don't forget to include a control of cells that are not expressing the protein, so you can exclude nonspecific interaction with another nuclear, or wherever localization protein.
I hope this helps, let me know if you have any further quiestion.
Best luck
If you are sure there were no technical problems with your WB I guess your antibody works well in IF but does not work in WB. Therefore, in order to see a band in WB, you should find an Ab recognizing a linear epitope of your protein. It is not easy to find antibodies working both in Western blot and Immunocytochemistry assays.
1) It is not uncommon for antibodies to recognize a 3 dimensional epitope that depends on the structure of the protein and not a linear sequence. As a result you will not be able to use these antibodies for western analysis. As others above have suggested, you'll need to get a different antibody for wester.
2) Also, are you sure your cells actually express you protein of interest? Can you knock it down and see the IF signal disappear. Unfortunately, many antibodies are not specific for a protein of interest.
Hi there,
IF is indeed more sensitive than WB as it allows protein detection within a single cell whereas one can't imagine loading the protein content of one single cell for WB, detection being at the microscopic level for IF.
Hi there,
I think Immunofluorescent is best way to show localization of specific proteins, but when we talked about protein levels, western blot should be more sensitive and reliable than fluorescence intensity quantification.
To add to the preceding answers:
You can also use a signal amplification system in IF, e.g. a tyramide-based system (TSA). You'd need a HRP-coupled antibody. This increases the sensitivity a lot (up to several hundred-fold) but you'd lose some resolution of the exact localization of the protein in the tissue/cells.
IF and WB had different properties. Generally speaking, IF had higher sensibility.
But if you want to detect the protein expression level, WB is higher. If you want to locate the specific protein's position, IF is better.
conformation-specific epitopes are indeed possible and could acccount for the apparent discrepancy. Do you have any info on the Ab? If it is raised against a short "linear"peptide, it is unlikely to be the case however. In any event, you should perform the appropriate controls (siRNA expts, control cell line etc...) to ensure your IF signal is specific.
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