dot blot - (Nov/10/2011 )

Hi all! I did a dot blot in a nitrocellulose membrane, 1 µL of the protein, denatured in Laemmli and 99 degrees for 5 minutes. Let it dry overnight. Then I blocked with milk 5% for 1 hour. Washed with TBST 2 times, 5 min. Incubated with the 1st antibody (diluted in store buffer). Washed same as before. Incubated with the 2nd antibody (diluted in Milk 1%). Then washed as before. Treated the membrane with ECL (1:1) for 1 minute and developed the film. The background was so high, that I could barely see the spots of the protein. Please, I need help...

I've had this happen a couple of times. Once our dried milk got spoiled so the blocking step wasn't working. Otherwise, there could just be a lot of non-specific binding of your primary antibody. If this is the case and the milk is fine, I would suggest adding 1% milk to your primary antibody solution and see where that gets you. Are you using any Tween or other detergent?

these are suggestions based on how I do dot blots and may or may not be the culprit of your high background
1. It is probably unnecessary to dry your membrane overnight, but this probably isn't accounting for the high background.
2. I don't wash in between blocking and primary antibody
3. I dilute both primary and secondary antibodies in my blocking buffer (5% milk in TBST)
4. I wash 3x10 minutes in between primary and secondary antibodies as well as after secondary antibody incubation with TBS.
5. The final wash after my secondary antibody treatment before development is with TBS (no T).
I hope these suggestions help!

You're probably right but this is the only one I have not tried myself.

If I'm in a hurry (and I almost always am), I don't either.

Should work.

3x5 min for me. Always works. I mean if we do 3x a couple of seconds in ELISA, I don't see why we should do 3x? MINUTES in Western blot.

Try TBST.
However, based on your post, you should be fine. I have cut corners on most steps in dot and Western blot and it's pretty robust. What vendor did you get your primary and secondary from?
Sorry for the lack of ideas...
Miha

Miha,
I am not having issues with my dot blots. Those were the suggestions I had for fabipp to reduce their background.

Ohh, sorry about that. I am however concerned about your suggestion about drying the membranes overnight. I would absolutely not suggest that because they become very brittle.

Miha, please actually read the posts before replying. I did not suggest to dry the membranes overnight. In fact, I said it is unnecessary to dry them overnight. However, considering that nitrocellulose membranes, which is what the original poster is using, are kept dry until blocking, allowing the spots to dry overnight will not cause the membrane to be brittle. Furthermore, that would be an entirely different problem from high background, which is the issue at hand.

Sorry kfunk106. You are quite right.