Triton X-100 and endocytosis vesicles - (Jul/25/2007 )

Hi!

I have some problems with lysis buffer for proteins. I am studing epidermal growth factor receptor (EGFR) so I thought that using triton x-100 into the buffer I could solubilize plasmatic membrane and then solubilize EGFR. The problem is when I treat the cells with EGF, the ligand of EGFR, I observe that EGFR disappears in total lysate at 5 min and later it appears again at 20 min!! It can not be a proteolysis and synthesis de novo process (it is happening too fast). It has been decribed that when EGF binds EGFR there is an endocytosis process so I think it's normal to observe a decrease in EGFR levels but it would mean that endocytosis vesicles are not broken, wouldnt it? What is it happening? What can I do to broke endocytosis vesicles as well? Some sugerence???
Thank you very much for your time!!

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That sounds weird. Actually, eventhough the EGFR is indeed endocytosed when stimulated with EGF, that does not mean that it will disappear from the total cell lysate. It is TOTAL cell lysate, you see, so it includes the endosomes, vesicles... unless you separate these from the lysate, and in that case, the lysate is no longer total lysate. Properly doing, EGFR will only disappear from the lysate when it has been degraded in the lysosomes, and that take hours. So, in your cases, there should be NO difference in the total EGFR level between 0, 5, or 20min. How did you make your total cell lysate? Do you have any control and what happened to them?

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How did you estimate EGFR presence in lysate? May be here cause of your weird results?

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Hi!!
Yes, I know it sounds very strange, so I'm still wondering what is happening
Almasy, I observe that EGFR level dissapears at 2 min after treatment with EGF and, like you, I think it is not enough time for degradation into the lysosomes. I use a "lysis buffer" to make total cell lysates wich has Tris, NaCl, EDTA, orthovanadate, PMSF, trypsin inhibitor and TRITON X-100 (1%). I add 200 ul lysis buffer to cells, scrape on ice, and incubate for 30 min at 4ºC. Then cells are centrifuged for 20 min at 4ºC and supernat used for western blot.
As a control I have cells that has not been treated with EGF (time 0).
After densitometry, the arbitrary units are:

Time with EGF (arbitrary units): 0' (1) 2' (0) 5' (0.2) 15' (0.7) 20' (1)

Circlepoint, I just stimate EGFR levels in total lysate by western blot, using a monoclonal antibody against EGFR from Transduction.

I am looking into the possibility of using another detergent such as CHAPS or NP-40. Well, I think I'm very lost...

Thank you very much for your time!!

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A few more things:
- Your Ab recognizes all EGFR or only either phosphorylated or unphosphorylated?
- You don't have loading control, like actin, tubulin... and haven't normalized your results against them?

I don't think that it is due to TritonX-100. We have done similar works with EGFR and don't have this problem.

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I also do not think that it has sth to do with triton x-100 if conc is enough; EGF seems to stimulate turnover of EGF-R which includes phosphorylation but may be glycosylation and/or ubiquitinilation? are there shifts in size of EGF-R? PTM may interefere with antibody binding even for non-phospho EGF-R Ab;

try to follow time-dependent localization of EGF-R with immunocytochemistry; use also alternative Ab´s including anti-phospho EGF-R

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