Hi Geraldine, If I understand your question, you are concerned that when the primary IgG is bound to several secondary labelled IgG, this may restrict access for additional primary?  The Yung/Seger antibody detects doubly phosphorylated ERK but does not bind to singly Ped one so the stochiometry is 1:1, there should be no steric hindrance between two IgG trying to bind nearby Ped sites. For quantification, especially on whole embryo, I would suggest to label your primary directly. The secondary IgG may introduce non-linearity, compounded by depth diffusion. I hope this helps.     

The problem of steric hindrance can be use of too high a primary antibody concentration.  If you look at the attached paper, you will see that for some antibodies too much primary gives quenching of signal (this can occur for chromagen or for fluorescence).  It appears to result from molecule crowding.  Direct labeling of a primary requires high titer and large enough volumes of antibody and is often not possible with commercial antibodies due to predilution by the company.  If one conducts a thorough titration of antibody with ABC methods, one can easily find a primary antibody range in which fewer antibody molecules bound to the tissue gives a linear and reliable reduction in signal.  working in that range on experimental tissue will enable quantification.  Over saturation will lose the linearity so the "right" range in critical.  I use the concentration that gives high standard deviation of intensity with no background (see Fos pictures in this paper).  The depth issue can be overcome by use of the correct dilution and long incubation times with the primary (72 hr, for example).

working in that range on experimental tissue will enable quantification.  Over saturation will lose the linearity so the "right" range in critical.  I use the concentration that gives high standard deviation of intensity with no background (see Fos pictures in this paper).