BAD blots - so many problems they don´t fit in a title (Mar/11/2011 )

Hi!

I´ve had several problems with my western blots. I attach a picture of the latest. Has anyone any idea what could be the cause of this kind of blot?

What I do to the blots: blocking 3%BSA in TBS-T0,005%, primary antibody overnight 4C agitation, washes always in TBS-T0,005%, secondary antibody 2h in RT agitation, washes, luminol 5min. The antibodies are incubated in a sealed blastic bag and washes and blocking are done in a plastic box.

My signal is usually very low for most of the antibodies I´m using, even to the "good" ones. The signal dissappears very quickly: usually I can only see bands in 1 min, after that the signal starts to fade away, I use the "add content of previous images the next image" when revealing but I cant get enhanced signal. Bands are often uneven, I cant count the results from them... argh, I´m getting mad with this, nothing works. In fluorescence I have had very nice, clear and clean images. The idea would be to detect total protein with fluorescence and phosphorylated with chemiluminescence in the same blot.

If the attached image rings any bell what might be the problem I appreciate ANY help!
Thanks and relaxing weekend!

Hi, here are some tips that may help your resolution.

- Wash with more stringency. I use 0.1% Tween in TBS.

- Try blocking with 5% milk, or increase BSA at 5%.

- Maybe your luminol/films are getting old. Can you try other solutions?

As for the technical part, i still do old school, hand-revealing westerns. So i can't help for any software related content.

Hi,

your fig. seem much background.
I often got these result. To reduce the background, I increase more time for washing in 2nd antibody 10 min for 3 times.
and I have to get rid of the excess ECL reagent by dipping with tissue paper.


For short signal. we use the better ECL such as ECL plus or ECL prime.

You should freshly prepared TBST and every buffer.

good luck