6. How do I avoid "splotchy" or uneven Western Blots?

6. How do I avoid "splotchy" or uneven Western Blots?

To avoid "splotchy" or uneven blots when performing a Western Blot analysis:
a. Extend the blocking time to optimize blocking effectiveness.
b. Extend wash cycles (this is particularly important for tissue homogenate samples).
c. Make sure that milk powder is in solution completely before adding it to the blot.
d. Spin the tube containing the antibody solution before removing some for use, in case of protein aggregates.

Antibody
Antibody
Western blotting / WB Antibody
Wstern Blot / WB Technique Center+
- Western blot introduction
- Western Blot / WB tips
- 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
- Western Blot / WB trouble shooting