Assuming your rabbit HRP is fine (other members used it successfully) you (as you probably guessed yourself) probably have a problem with your primary and the datasheet is surely consistent with that (concentration unknown, not affinity purified etc..); the pics look good though...  

That being said and as suggested above I would try a secondary alone (should be clean as a whistle at your "default" exposure settings) just to be sure; are you using the same kind of membrane (PVDF, NC etc..) as the others were?

If both milk and BSA give you problem as you mention, move away from the cow!  Your  primary Ab or secondary may cross-react with bovine proteins.  Try gelatin or non-protein blockers (PEG etc..).

Could also be that you used too much primary/secondary and that you are saturating your system somehow but it seems less likely based on your description. Good luck!

sounds like your secondary rabbit antibody is binding to the blocker. Use a different blocker or different secondary antibody. you can easily test this, block the membrane only then incubate with secondary only. If blot is not black then potentially primary antibody problem.

You could reduce time of incubation with antibodies and increase stringency of washing steps or include a more stringent buffer with the antibodies and use a more dilute antibody solution

bw

Mandy: We had all kinds of non-specific troubles with the traditional WB system: a primary Ab followed by an anti-Ab-HRP.  Instead, we conjugated all of our primary Abs to HRP, and avoided the anti-Ab-HRP.  The results were remarkably beautiful even with straight serum samples were analysed.  Good luck.

Assuming your rabbit HRP is fine (other members used it successfully) you (as you probably guessed yourself) probably have a problem with your primary and the datasheet is surely consistent with that (concentration unknown, not affinity purified etc..); the pics look good though...  

That being said and as suggested above I would try a secondary alone (should be clean as a whistle at your "default" exposure settings) just to be sure; are you using the same kind of membrane (PVDF, NC etc..) as the others were?

If both milk and BSA give you problem as you mention, move away from the cow!  Your  primary Ab or secondary may cross-react with bovine proteins.  Try gelatin or non-protein blockers (PEG etc..).

Could also be that you used too much primary/secondary and that you are saturating your system somehow but it seems less likely based on your description. Good luck!

Thank you for your answers! One of the annoying things is that the primary Ab is stored in the -20 and was used by another lab member in April of this year, and has been aliquoted out to avoid freeze-thaw. The Ab worked perfectly fine for his westerns, and he blocked with milk and used the same membrane as I am using. Is it possible for Ab's to go bad in 6 months when stored in the -20? I've never experienced that. 

The secondary is used by other lab members every day with no problem, using the same membranes. 

Assuming we're dealing with a research freezer (stable -20) and your Ab is in a proper buffer (concentrated with glycerol (or even stably frozen in aqueous buffers)), it should be stable for way longer than 6 months; Unlikely due to a dead Ab, as you seem to have too much signal...not too little.  Did you talk to the lab member?  Are you sure those are the right aliquots?

Are you by any chance drying your blots?  For nitrocellulose, that's a big no-no in my experience as background can become a significant problem.

sounds a little confusing to me if others have had no problem with the antibodies, like background. The only other suggestion is that your buffers maybe contaminated. Make fresh everything (or even borrow some buffer from those not affected by background)

Primary antibody and Secondary antibody source should not be the same animals. Check your antibodies. You might need to check your steps and reagents used for blocking unspecific binding procedures too.  Also check your final step of western blot in which you used reagent to developed your bands (your reagent dilution if it is correct or contaminated or etc)

I think the difference is in the quality of the antibody you are using. If I were you I would probe the membrane only with rabbit-HRP Ab (omitting the primary) and then develop with ECL. If the background is still high you should change the secondary Ab for a better one. If it is good, you should change the primary Ab for a new one. I do not think buffers and blocking reagents are responsible for such high background, since they work well with mouse Ab pairs. 

You can shorten ECL exposure time as well as use more diluted ECL You can also use protein free blocking reagent like (Nacalai Tasque company,Japan). You can cover up ECL exposure material with plastic rapping paper or other plastic to avoid light during ECL incubation time..and use all fresh material including gel ..hope you will get better result.

Try to reduce time of exposure, if this does not help even with 10sec exposure etc... try to change your ECl reagent.. if possible.. use a weaker ECL reagent.. I experienced same once.. I switched from  Pierce ECL to that of Amersham ecl reagent, and this helped.. also you can increase the concentration of detergent in your wash buffer.. eg if using 1%.. try 1.5 or 2% and bit longer washings...

happy working :)

Thanks all! To answer your questions: 

The lab member is gone so I can't ask him, and since he took very, very poor notes, I don't actually know what he used as a blocking agent. I agree that the Ab should be stable. But, I've since used a new primary antibody, and it still does the same thing. 

As for the secondary potentially being an issue, I've cut my blot in half and used another rabbit primary on the left along with my rabbit Cas9, and that developed completely fine. I even tried switching to the rabbit fragment secondary to no avail.

I definitely don't dry out my blots, although they are transferred with an iBlot dry transfer system. After that though they go right into block and are never dried again. It has to be something with the primary antibody though, because like I said, I split my blot in half and did everything identical to the other half that I did to my Cas9 and the other half turned out fine. 

Is it possible that something in the primary is binding to the 5% milk and that's why it's all over the place? 

Hi Mandy,

Did you finally found the reason of your black western? I have the same problem now.

Gisela Gerardi No I never did find out, I switched to using the WES system instead and it works fine. The WES is a digital capillary system.