I am currently having issues with my immunoblotting which I feel can be resolved by using an alternative blocking buffer/agent. There appear to be numerous blocking agents available and I wanted to enquire which would be best for my application.
To summarize, I am performing Western blotting analysis to look at markers of ER stress in diabetes using tissue from a mouse model. However, using the current combination of antibodies (primary raised in rabbit, secondary raised in goat) with either BSA or milk, detect 2 strong non-specific bands which appear to be the heavy and light chain of IgG (I see the same pattern when I use secondary alone with not primary). Can anyone recommend a blocking buffer that I can use which will eliminate this 'non-specificity' as these bands are interfering with my protein of interest?
If what you say is true, then you just need to find a secondary antibody that is more species-specific. There are many anti-rabbit IgG secondary antibodies that will not cross react with mouse immunoglobulin.
In addition to what Mark said to try a more specific anti-rabbit secondary antibody, perhaps mouse pre absorbed if you can find one. There's another possibility if you're running normal reducing gels. There are a number of specific secondary antibodies that only detect native primary antibodies, basically no reduced/denatured IgG from your gel, only what you probed the blot with. They have been developed for immunoprecipitation because you'll have a lot of Igs in IP samples and even with our regular very specific 2nd anti-rabbit Ab I pick up the mouse Igs from my IP, so the anti-native 2nd Ab is much better.
You can get small samples of the abcam VeriBlot Ab for 130€ or Sigma have some as well.
We incubate the antibodies in TBS with 5% milk, 0.2% tween-20 and wash with the same buffer containing 0.8M NaCl. Adding NaCl worked nicely in our hands to remove non-specific binding.
We incubate the primary antibodies in TBS 0.2Tween-20, 5% milk and we wash in TBS 0.2Tween-20, 5% milk containing 0.8M NaCl. The NaCl helps to reduced non specific binding and it worked well in our hands.
hm, there is not IP anywhere. i would not think that a diabetic model containes such an amount of IgG (mouse). mouse blood can cause some problem, if that tissue contaminated a bit or more. even stressing can generate your extra signal. try a blot without secondary, probably you got an extra "enzymatic activity" (hemoglobin gives a signal in chemiluminescence even) .
Thank you for all the very helpful suggestions.
Like Mark and Kerstin have suggested, I'm going to test an alternate secondary (VeriBlot Secondary Ab from Abcam) as I feel it will resolve the issue (pun intended!).
Melanie- Thank you for the advice of using NaCl to reduce non-specific binding. Are all your washes performed using NaCl in the wash buffer?
Kalpana- I'm confident that it is not the primary as I've optimize the antibodies with various combinations of BSA/Milk, and Tween with the inclusion of positive controls beforehand. I've not tried testing the antibody using non-reducing conditions but it will be something I will consider in the future.
Attila - I'm working with sciatic nerve isolated from diabetic mice where we have identified a >7 fold increase in Ighg1 suggesting that what I'm seeing in WB is heavy and light chain IgG.
Thanks again
Hi
In terms of interfering heavy and light chain on the blot, a very good alternative is to use the TrueBlot secondary-HRP antibodies in combination with your primary detection Ab. The TrueBlot Ab will not recognize heavy or light chain on your blot. Works very well.
thanks ketil
- Badges
- Science method