Both of the concentrations seem rather high to me. Of course it depends on the recommendation of the manufacturer, but the primary should be around 1:500-1000 and secondary in the order of thousands to something like 25 000.

The concentration also depends on system you use for development. I remember I was working with some solutions from Roche and they had a table, where they showed suggested concentrations for several of their kits.

Do you have any positive control for these antibodies?

Hi Victoria,

I agree on Tomas comment. 

As an add-on, it might be due to blocking process, as the image seem to be 'dirty'. Are you using skimmed milk? How long have you blocked the blot? 

Hi there,

To go further with blocking issue : I use 3% non fat milk in TBST(0.1%) for at least 30-60 minutes and all the antibody dilutions are made in the blocking solution.

This clearly looks to me to be a problem with blocking, that needs to be addressed before optimizing the concentration of primary and secondary antibodies. The use of BLOTTO (which includes milk solids and detergent) is common and works for many antibodies. But every antibody/serum is different so you will likely need to try different blocking reagents. Another issue that may prove important is the filter: nitrocellulose vs PVDF. You will get very different results and need to optimize for your protein of interest and antibody used. And Dominique makes a good point. We use BLOTTO for blocking but also use it for primary and secondary antibody incubations. Finally, after the secondary antibody make sure you wash well to remove the BLOTTO and any residual antibodies or proteins. These too can add to background. We use at least three 10-15 min washes in TBS with Tween 20 (0.1%) washes, prior to developing ECL.

Thanks for the answers.

I'm using nitrocellulose membranes. The blocking solution is 5% non fat milk in TBST(0.1%). Normally, I block the membrane for 1 hour at RT. I've attached a picture of the b-actin protein, so you can see there is no problem about the blocking process. Anyway, I'm repeating the assay after a stripping strep and I've modified the blocking time (2hours) and the primary and secondary ab dilutions. Hope it works.

Let me guess, this actin blot was developed up to 1 minute, while the first ones were developed for several (tens of) minutes?

Not at all Tomás. The image acquisition lasted the same for both proteins. It was incremental exposition with 10s intervals and no more than 2 minutes were needed for those results. Any suggestion? Thank you!

Nice looking actin control. This actin antibody has been used a control likely by many labs precisely because it gives such nice results. Every antibody/antiserum is different. Several of our rabbit polyclonal antisera give ugly blots like the one you first showed. This may well be the result of contaminants in the serum, which could be eliminated by purifying IgGs on protein A/G Sepharose beads. Or simpler you could try ammonium sulfate precipitation of IgG's followed by dialysis to clean up the serum. Or you could be unlucky and it is your antibody that gives high background, in which case either keep trying different filters and blockers or get a new antibody. Sorry but that has been our experiences with almost 100 different sera.

Thank you Richard, very useful information and observing the results it's very likely that this antibody it's the problem. 

Victoria, I have had the same kind of problems with some of my blots.  It is most likely your secondary.  Have you used this secondary before with good results?  

One of the times it was a problem with a secondary that I got from Santa Cruz.  They even sent me a new batch and I had the same problem.  I have had other problems with some of their other products, too.  I try not to use that company any more.

No, the secondary ab is fine because I've used with other proteins and the results were clear. As you say, the sirtuin ab it's from Santa Cruz and I have the same feeling as well. Thanks for your care! 

Hi Victoria,

I also think the problem comes from the primary ab. Just one thing, the speckles most probaly arise through precipitates of the primary antibody. Filtration or spinning the precipitates down would do the trick to loose the speckles, but I still would suggest to try another primary antibody.

Best,

Carsten

Thank you Carsten for your suggestion, that could mend the problem :). But yes, another primary antibody would be the best solution.

Thank you all for the contributions!!