I'm posting something for the first time in this forum though I have learned a lot from others' posts. I am trying to understand whether chemoattractants and/or leukocyte adhesion to endothelial cells phosphorylate my target protein in endothelial cells. After optimizing several things, I have got my desired band. But the problem is I don't know how I can control my loading. I have different treatment groups (chemoattractants and stimulated leukocytes esp. neutrophil). In chemoattract groups I have only endothelial cells and I can use beta actin or other internal controls but in stimulated leukocyte treated groups there are two types of cells endothelial cells plus adherent leukocytes. So, there is more beta actin and total protein in leukocyte treated groups. How can I compare chemoattractant and leukocyte treated groups. Only thing I can control and in fact I am controlling is the number of endothelial cells. Neutrophils that I am using are deficient of my target protein. Now I am feeling helpless. Any suggestions? Thanks.