4. Why does my antibody activity diminish in Western Blot over time?

4. Why does my antibody activity diminish in Western Blot over time?

If you are using one of our antibodies for Western Blot analysis and the reactivity seems to be diminishing over time, some points to consider are: a. Different samples were used - cells in culture will change characteristics over time, so we suggest thawing fresh cells.
b. Samples degraded in storage or from repeated freeze-thawing.
c. Antibody was contaminated - spin down vial and check for precipitate.
d. Second step reagents are not working.
e. Protein transfer was inefficient - stain blot with India Ink after transfer to confirm transfer of protein from gel to blot. Please note that antibodies are very stable and are unlikely to "go off" over time when stored appropriately.

Antibody
Antibody
Western blotting / WB Antibody
Western 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