2. Why is there a weak signal or no signal at all?

2. Why is there a weak signal or no signal at all?

Several variables may account for this:
a.This may be due to inadequate transfer of proteins during electroblotting. Try staining the membrane with India Ink or Ponceau-S immediately after the transfer. This is an effective and non-interfering method to verify proper protein transfer. Consult our recommended Western blotting protocols to optimize these electrophoresis and transfer conditions.
b. The detection enzyme may be inactivated. Sodium azide inactivates horseradish peroxidase (HRP) irreversibly, so do not include sodium azide in any HRP-labeled reagents. Bacterial contamination also diminishes HRP activity. HRP conjugates should be kept bacteria-free, handled with sterile technique and stored under recommended conditions.
c.The polyvinyl wrap from certain sources may quench the signal. We suggest repeating the incubation with chemiluminescence reagents and placing the blot between two pieces of write-on acetate transparency film, and then expose the film.
d.The expression of the protein of interest may be very low. Sensitivity may be increased by performing an immunoprecipitation prior to the Western blot.
e.In cases of very weak Ag expression, eliminating Tween during primary Ab incubation may improve Ab binding.
f.To better facilitate Ag-Ab interaction you could incubate primary Ab at 37°C with gentle incubation and further incubate it overnight at 4°C.

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