I have to quantify 3 different target proteins. but each time I do wetern blot, I get a very distinct and sharp nonspecific band above 100kD.
I have tried:
1. different chemicals form different vendors,
2. Primary antibodies obviously different
3. blocking both with BSA or milk
4. vectastatin secondary antibody (two anti rabbit, one anti mouse), avidin biotin complex-HRP conjugated (this is common for all runs)
5. developed with DAB (common for all runs)
6. even tried different tissues like brain tissue, retina, spinal cord
7. INTERSTING is that band appears same intense in all my 9 groups that is independent of total protein concentration (as ihave tried loading total protein with large differences).
What could be your opinion?
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