just a word of advice - better to lean from others mistakes. ECL and missing bands (Jun/05/2006 )

hello,

i just wanted to impart a little story that happened last week.

i was doing some western blots. everything was working well. good gels, good transfer, lovely strong signals using an ECL kit. but then it all went horribly wrong.

the gels were good. there was good transfer. but then there were no bands showing up on film.

first, i thought it was the primary antibody. so i got a new batch out. still no bands.

so, i figured, it must be the cell lystates. made a new batch. still no bands.

so, it must secondary antibody. still no bands.

took a weekend off, and saw a movie.

went back to work, and someone else in my lab wasn't getting bands either. hmmmm. he thought it was the primary antibodies.

we finally ran out of the ECL kit, and opened up the new one.

finally, we see bands.

what happened, apparently, was this:

the ECL kit has two bottles. the solutions in each are not supposed to come in contact with eachother, until it comes time to actually use it.

someone had probably taken a little bit of solution 1, and with the same pipette, taken a little bit of solution 2. the residual amount left on the pipette tip had contaminated the solutions, and made them not work.

we wasted 2 weeks trying to figure out what went wrong.

learn from us. ECL kits are antisocial.

vetticus