Wrong Secondary Ab and still get a signal? - How to explain signal from wrong Ab? (Jul/24/2004 )

I am trying to understand how I got a clear ECL signal on my Western Blot membrane, even after discovering that I used the wrong secondary Antibody.

This was a repeat experiment, but we had to change to a different brand of primary antibody (ran out of our original in the middle of the night - you know how that goes). Our first version of this primary was raised in mouse, but I did not realize at the time that the new antibody was raised in rabbit instead.

After incubating O/N with this new primary, I washed it and incubated it with a mouse secondary antibody and carried out the rest of the development procedure. In the end, we did have to use an intensifier on the membrane (which is what got me to realize I'd forgotten to check where the primary was raised), but the signal on this repeat experiment was identical to our first trial with the same cell samples/ concentrations.

I am wondering if this new ECL signal should be questioned, because we used the wrong secondary Ab (we should have used anti-rabbit and instead used anti-mouse), if there is a way that the anti-mouse secondary really did bind with the rabbit-raised primary, or if the primary signal and concentration (1:100 ratio, as recommended by the company) was so great that we just did not need a secondary?

Thank you for your insight!

Immunoglobulin staining due to crossbinding of secondary antibody to another specise is not uncommon.

yeah, postdoc's right!

crossreactivity to some extent is quite common...
so no need to worry!

I don't quite understand your point with the primary antibody....
I thought, the primary antibody would not carry the enzyme you'd need for detection, hence the need for a second, labeled antibody - right? So the the primary antibody alone can never give you a signal, regardless of how much Ab is binding to the membrane....

maybe i misunderstood something?

Mike