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Do antibody solutions settle? - (Nov/08/2007 )

This may sound really stupid but when I went to develop my western I got really really weak signals from proteins that we always get really strong signals for. I realize now that I didnt vortex the secondary antibodies before I added them to my membranes. Do you think this could have caused my super weak signals? I"m adding more, vortexed secondary and trying again, but just wanted to see if this ever happened to anybody else.

-yes, protein is DEF on the blot, PonceauS looks great
-Blocking 5% BSA 1 hour RT
-Primary O/N
-Secondary 2 hours RT 1:10,000 (10ug/ul)
-Pierce Femto Development kit


yes. proteins do not form true solutions. they form suspensions and, while they are relatively stable, they will drop out.

unless your protein is very fragile you should always mix before using.


That pretty much solved the problem. I vortexed my secondaries before adding again and the blots developed fine.