I am doing western blot for my samples and every-time getting high background with multiple nonspecific white bands, even marker gets stained as white bands. I have tried all different parameters to troubleshoot this issue, like low protein concentration, blocking time, washing time, different antibody dilutions but every-time it gives same result. I am using Clarity Western ECL Substrate, # 170-5060, Biorad. It would be great if anyone can suggest solution for this?
A few questions back to you:
1) do you see bands where you'd expect them to be, along with background? The background looks like what you'd see if you just stained with Coomassie, suggesting that you are not really binding to your antigen.
2) are you exposing the film for a very long time (relative to other blots done before)? If your primary isn't working, but you use a really long exposure time, you will invariably see non-specific binding similar to what you're seeing.
3) Is your marker designed for Western blotting? You may be using a marker that is designed to show up on a blot.
As my questions suggest, I believe your problem is that you're not effectively probing your target antigen, and all you're seeing is background. This could be for several reasons: 1) you didn't load enough sample to detect your (low abundant) target; 2) your primary antibody is bad; 3) your secondary antibody is bad or not matched to the primary; 4) your blocking agent is interfering with primary binding to the antigen. If you saved the membrane, try washing it, blocking again, and probing with a different primary to an antigen that you know should work well (like a load control).
A few questions back to you:
1) do you see bands where you'd expect them to be, along with background? The background looks like what you'd see if you just stained with Coomassie, suggesting that you are not really binding to your antigen.
2) are you exposing the film for a very long time (relative to other blots done before)? If your primary isn't working, but you use a really long exposure time, you will invariably see non-specific binding similar to what you're seeing.
3) Is your marker designed for Western blotting? You may be using a marker that is designed to show up on a blot.
As my questions suggest, I believe your problem is that you're not effectively probing your target antigen, and all you're seeing is background. This could be for several reasons: 1) you didn't load enough sample to detect your (low abundant) target; 2) your primary antibody is bad; 3) your secondary antibody is bad or not matched to the primary; 4) your blocking agent is interfering with primary binding to the antigen. If you saved the membrane, try washing it, blocking again, and probing with a different primary to an antigen that you know should work well (like a load control).
1. If you use a secondary labeled Ab, it is recommended that expose the blot paper just with the sec Ab without adding primary Ab. After visualization with ECL, if you see bonds as well as before the problem will be with the sec Ab. You may need to dilute the sec Ab more and if the problem existed yet replace it with another.
2. Also I recommend to check the purity of water you used for preparing buffers and reagents.
Thank you so much Andrew and Mohammad for your inputs. Wrt questions:
1. Yes, we do get our band of interest but it’s visible In negative control as well.
2. Exposure time is nit for longer period we do manual for few seconds and then go for auto mode
3. Marker is designed for WB
4. We had tried different concentrations of protein but same result
5. Blocking reagent also tried different.
All buffers prepared freshly with purified water
6. Have tried diluted sec ab as well nd it works well when we probe w other primary antibodies.
7. And when the same blot was reprobed with different primary ab it gave excellent blot results.
so we suspected may be primary ab is not reactions at all.
Neha, I'd conclude the same. Your primary ab is bad. If it is brand new and had been shown by the vendor to work for WB on your species, then you might be able to return it for credit. If it was an older ab, then it could have been damaged from poor storage, insufficient protection against freeze/thaw, or it is just too old. Good luck!
Thanks Andrew.. thanks alot. Ab is brand new, as it didn’t work out so company replaced it but with that also it didn’t work. So ll try out other options.
regards
neha
I think the problem is confound to secondary Ab brand, dilution, and exposure time during imaging ur blots. Plz try to dilute it about 10 fold of the current dilution, lessens the incubation and exposure time. If u get faint bands with clear background u can increase the conc of first Ab a little bit. Plz also use new washing solutions. Good luck
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