Hey, I did WB and IF in different time but with the same cell line, however, the results from WB and IF seem incompatible. WB and IF both show the protein expression level right? How could they be different?
One of the possibilities is I did WB and IF in different time so the expression of the target gene might shift according to the cell cycle. Is there any other possibility?
Hi Yang Wu,
I assume WB stand for western blotting .. but could you define precisely what IF stands for please? Isoelectric Focusing ? ImmunoFluorescence ? Could you please a bit more what you actually did ? Many thanks ?
Philippe
Hi Yang,
Can you clarify which technique IF specifically refers to here?
It's possible both are accurate, but if say you're looking at Immuno Fluorescence staining and a western blot you're looking at somewhat different things and in different ways. IF you'd be looking at cells individually, westerns you're looking at the average of all harvested cells pooled together. What is the half-life of your protein? How far apart were the time points of the experiments (1 hr, 2 hr, 12 hr, etc?)? Were these cells synchronized in any way so that you would expect for them to be at a specific time point in the cell cycle and all cells would be in the same phase? How many technical and/or biological replicates have you tried this with?
Thanks!
Dear Yang,
in principal the IF and WB should show the same results (at least with regard to fold change, e.g. like finding in both assays 3-4 fold up/down). Did you include suitable positive and negative controls in your setup? Do these behave as expected or is there something odd as well?
The following reasons for different results between IF and WB might be considered:
-different time points of the assays (as you mentioned), try to do the experiments next time in parallel to decrease variability
-cell cycle-dependent expression changes (you could try to synchronize your cells) could play a role but can be avoided by running the experiments in parallel
-different antibodies were used for the two assays?
-normalization of western blot signal (it might be worth to look at two or more housekeeping genes to see which one is not changed in your specific setup)
-Your protein of interest might be lost/destroyed during WB sample preparation
-fixation of samples for IF could "mask" or destroy the antigen (check if the antibody is compatible with your fixation)
-the normalization methods for IF results are sometimes very tricky
If you can still not make sense out of the data after analyzing several replicates you could try an additional method like FACS.
However, more information about the experimental details could help to identify potential problems.
Best,
Marcel
It is not uncommon for IF and WB to yield different results since (among other things) both approaches present different epitopes to your Ab (native vs non-native) and typically, different buffer systems. Western blot is in principle easier to control for since size is an important parameter. However in my experience several suppliers will sell you "working" Abs based on size selection alone, which is a big problem. In IF cellular localization (if known) can help determine if the signal is the right one. in any case, as mentioned earlier, you should have appropriate controls (positive and especially, negative (e.g. siRNA)).
Good luck!
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