Crazy: ab burnout or something? Higher quant had lower signal? - (Jul/24/2009 )

This is weird. I had an extra lane on my Gel so right next to one of my samples I added 3x the ug of protein of the same sample. So sample 1-100%, 2-100%... 4-100%, 4-300%,...

Now on the western I cut the membrane in half and probed for Actin on the bottom and NRSF on the top. The bands all look nice, the 4-100% versus the 4-300% looks like it should for actin.

But the NRSF band for 4-300% is WAY lower signal than the 4-100%? What the!? What am I missing here, how is that even possible?

Any ideas? How can I trust my WB?

What detection method did you use? Fluorescent or ECL?

Your guess was probably right. You probably overexposed the fourth lane because it was overloaded with protein (and therefore had too much label). This is especially common with ECL methods.