Dear all, I did the immunofluorescence for Hep3B cells.
Cells were fixed by 4% paraformaldehyde 10min and permeabilized by 0.1% TRITON-X100 10min, blocked by 1% BSA 30min and incubated by mouse-monoclonal Antibody EGFR ab30 overnight, Alexa-555 anti-mouse antibody, post-fixed by 1% paraformaldehyde 10 min, incubated by DAPI 100 ng/ml.
The cells were starved for 16h and then stimulated by EGFR ligand ( amphiregulin) for 10min or not treated. However, both the cells showed that EGFR were distributed everywhere in the cells. Theoretically, EGFR is membrane protein and should be located in membrane. I don't know why.
Hi Liu
if you mean HER1, I wouldn't surprised since the EGFR family can translocate to the nucleus and could act as transcription factor. the only things I am concern about it you said that you starved your cells, as you know the stress factor could activate many of signal pathway so I would think carefully, and I would use confocal microscopy since its more accurate. and I would use controls as many as you can to test the specificity of your primary and secondary Ab.
best wishes
In addition, other possibility, is that EGFR can be cleaved by ectodomain shedding mechanism and then regulated intracellular proteolysis, therefore releasing IC and EC domains. In this way you can get a IC soluble fragment that could be giving you such signal. Of course this is in the case you are using an Ab against the IC domain. Are you? I attach a review on the subject that may be useful to further read about ES and RIP.
Before looking into the scientific aspects and possibilities of EGFR sgnaling, it will be important to make sure that you have specific signals. Normally expected pattern will be some membrane association and vesicular staining. Triton permeabilization could be harsh on membrane associated proteins. There is an abreview for AB30 with some protocol details ( http://www.abcam.com/egfr-antibody-egfr1-ab30.html ). May be it makes sense to give a try.
Hi, @Massar Alsamraae thanks for your answer. Even though EGFR can translocate to the nucleus and act as transcription factor, after the ligand stimulation they distribute always in cytoplasme, but not recruit to plasma membrane. This is the point that I can not understand. I starved the cells because I want to see the effect of EGF ligand stimulation without the influence of growth factor of serum in medium. As to the antibody specificity, I did the control experience that I added only secondary antibody and there was no signal.
Thanks again!
Sincerely
Dear Liu, by specific signals, I mean ruling out the non-specific detection of something by your primary antibody under the particular staining conditions.
I know that many people include secondary-antibody only controls, but this only tells that your signals are primary antibody dependent. There are two additional possibilities of non-specificities which cannot be excluded by the above control-
1. the primary being non-specific and detecting some other proteins
2. primary antibody has enough specificity, but due to improper (not well suited) fixation methods, u have only some background signal and the original antigen is lost / mislocalized.
Normally u need knockdown / KO controls for specific detection of total proteins and some inhibitor / stimulation protocol to check for signaling events. In your case, since a working protocol was described for the same antibody, I would give a try with the same.
If you want to have a look at what difference small fixation/permeabilization changes can do to your signals, you can have a look at the abreviews which I submitted for this Anln antibody. In one case it was a granular non-specific pattern throughout the cells and in another one, there was specific signals as expected:
http://www.abcam.com/anillin-antibody-ab5910.html?productWallTab=Abreviews
Hope it helps: Manoj
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