unspecific bands all over wb

[missoliverita]

Hello to you all,

I dont have a lot of experience doing western blots and I received a protocol from my superior before she left. I did a test western blot with a couple of samples but unfortunately I got a lot of other bands all over my blot.

I blocked for ih in 5% milkPBS-0,1%Tween, washed 3x with PBS-Tween incubated with 1:500 dilution of anti-JunB antibody ON at 4°C , washed 3x times in approx 50 ml of PBS-tween then incubated with anti-mouse IgG for 1h at RT, washed again and developed.

Where could the problem be?
Ideas??

[danfive]

Problem could be:
1. Too much protein loaded
2. Antibody concentration too high
3. Antibody incubation time too long
4. Blocking step insufficient

I suspect #2 and 3.
You need to optimize. Try a short incubation with primary antibody 1 or 2 hours. And try dilutions 1:1000 and 1:2000.
BTW your secondary antibody dilution should be between 1:20,000 and 1:40,000 approx.

Good luck!

[missoliverita]

thanks!!!! Im rather careful about following the protocol given by my superiors but I have a membrane that I can strip and then ill try out more dilution!!!!!!

[missoliverita]

Uhm......i have a question though, surely the dilution is important but if my membrane is large and I use 5 ml endvolume its not the same as if I use 10ml endvolume is it? I mean my membranes would have to be the same size all the time for a dilution to always work the same, right? If I had a minimembrane and I used 10ml of diluted antibody that would still be too much.

Stupid question maybe but I was wondering

[danfive]

You should standardize--that means use same size gels and membranes, as well as same volumes, incubation times for your western blots--that way you will know your protocol works the same everytime (it's also eliminates variables when troubleshooting).