adhesion molecules by westerns - (Feb/28/2007 )

Hello,

my work involves isolating endothelial cells from the heart and lung of mice, and then characterising the adhesion molecule expression levels from them. the mice are p53 tumor suppressor mutants. I have had problems in doing my westerns. I see actin and p53 when i am doing my westerns, but I do not see the adhesion molecules. what could be the reason?/

Thank you

Pmaj

---

could be a general problem with immunoblot (some technical problems or not well working Ab);

or problems arise from preparation method; do you use proteases during the cell isolation process? may be extracellular domains are destroyed...

---

Are you sure that the cells you work with are the good ones? Because if you do not see the proteins and you ab are ok, maybe it's because the cells just don't express the adhesion molecules.

---

Hi there,

I do use proteases while isolating the proteins from the cells. Secondly, I think the cells might be the problem, since i do not see even the markers for the endothelial cells by westerns!!

Isolating the endothelial cells from the organs using magnetic beads has been a problem.
do you have any suggestions on the same.

thank you for your reply.

---