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Unknown bands (at 150 kDA) during western blotting of beta catenin?
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Laurie Keller
Unknown bands (at 150 kDA) during western blotting of beta catenin?
Hello Stefan,
It sounds like it's just non-specific binding of the antibody. You could try a different blocking solution (e.g. BSA instead of milk) to try to resolve this. Alternatively, try running a Western blot with your usual samples alongside a sample where your target protein was knocked-down. If you still see the bands after knock-down, they are definitely unspecific. Hope this helps and good luck.
It sounds like it's just non-specific binding of the antibody. You could try a different blocking solution (e.g. BSA instead of milk) to try to resolve this. Alternatively, try running a Western blot with your usual samples alongside a sample where your target protein was knocked-down. If you still see the bands after knock-down, they are definitely unspecific. Hope this helps and good luck.
NSB is the most likely explanation for unexpected bands in Western. As Monika suggested, changing the blocker often helps. You can increase the protein up to 5%, increase Tween to .5%, and try adding 0.1M glycine (before adjusting pH). If you have lots of money, Pierce and BioRad sell excellent commercial blocker cocktails. You might also see lower NSB on a PVDF membrane. And if you happen to be running native PAGE, which I reckon is unlikely, 150K can be IgG from serum in your medium.
NSB is the most likely explanation for unexpected bands in Western. As Monika suggested, changing the blocker often helps. You can increase the protein up to 5%, increase Tween to .5%, and try adding 0.1M glycine (before adjusting pH). If you have lots of money, Pierce and BioRad sell excellent commercial blocker cocktails. You might also see lower NSB on a PVDF membrane. And if you happen to be running native PAGE, which I reckon is unlikely, 150K can be IgG from serum in your medium.
Hello Stefan,
It sounds like it's just non-specific binding of the antibody. You could try a different blocking solution (e.g. BSA instead of milk) to try to resolve this. Alternatively, try running a Western blot with your usual samples alongside a sample where your target protein was knocked-down. If you still see the bands after knock-down, they are definitely unspecific.
Hope this helps and good luck.
Hello Stefan,
It sounds like it's just non-specific binding of the antibody. You could try a different blocking solution (e.g. BSA instead of milk) to try to resolve this. Alternatively, try running a Western blot with your usual samples alongside a sample where your target protein was knocked-down. If you still see the bands after knock-down, they are definitely unspecific.
Hope this helps and good luck.
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NSB is the most likely explanation for unexpected bands in Western. As Monika suggested, changing the blocker often helps. You can increase the protein up to 5%, increase Tween to .5%, and try adding 0.1M glycine (before adjusting pH). If you have lots of money, Pierce and BioRad sell excellent commercial blocker cocktails.
You might also see lower NSB on a PVDF membrane.
And if you happen to be running native PAGE, which I reckon is unlikely, 150K can be IgG from serum in your medium.
NSB is the most likely explanation for unexpected bands in Western. As Monika suggested, changing the blocker often helps. You can increase the protein up to 5%, increase Tween to .5%, and try adding 0.1M glycine (before adjusting pH). If you have lots of money, Pierce and BioRad sell excellent commercial blocker cocktails.
You might also see lower NSB on a PVDF membrane.
And if you happen to be running native PAGE, which I reckon is unlikely, 150K can be IgG from serum in your medium.
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