3. Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?

3. Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?

Check the blocking solution that you are using. Non-fat dry milk suit for all blots except phosphor tyrosine blots, which has Phosphorylated proteins that bind to the membrane. By blocking with this reagent, potential epitopes are present all over the blot. Diluting the anti-phosphor tyrosine antibody in this solution will occupy many of the epitopes, leaving fewer antibodies available for detection. We suggest to use 1% BSA in TBS + Tween 20 as the blocking solution - do not use milk.

Antibody
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Western blotting / WB Antibody
• Western Blot / WB Technique Center+
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- Western Blot / WB FAQ
1. Why are there multiple bands on my blot?
2. Why is there a weak signal or no signal at all?
3. Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?
4. Why does my antibody activity diminish in Western Blot over time?
5. How do I overcome inadequate or poor transfer problems?
6. How do I avoid "splotchy" or uneven Western Blots?
7. What can I use as positive control lysates for sinobiological WB antibodies?
8. Too many bands on a Western blot
9. No Signal or Weak Signal
10. Nonspecific Bands
11. High Background
12. How much protein should I analyse?
13. What percentage acrylamide gel should I use to resolve the proteins?
14. What membrane should I use?
15. What blocking buffer should I use?
16. What dilution of primary antibody should I use?
- Western Blot / WB protocol
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