9. No Signal or Weak Signal

9. No Signal or Weak Signal

Possible Cause & solution
a.The cell or tissue type does not express the protein of interest. Perform a positive control, preferably from cell or tissue lysate already verified to express the target protein.
b. Improper cell treatment. 
Stimulate cells with the appropriate chemical, protein, etc. Verify that the stimulation works.
c. Improper sample preparation for gel loading.
Ensure that the protein in the lysate is stable. Use appropriate protease and phosphatase inhibitors, etc. Ensure protein samples contain SDS, and have been heated prior to gel loading. Include a reducing agent such as dithiothreitol (DTT) and/or 2-mercaptoethanol.
d. Insufficient amount of antigen present.
Load more protein on gel. Also, if the specific antigen concentration is too low, try enriching the antigen by fractionation or by immunoprecipitation.
e. Proteins did not transfer properly to the membrane.
Wet PVDF membrane in methanol or nitrocellulose membrane in transfer buffer before use
Ensure there is good contact between the membrane and the gel
Optimize the transfer time
After transfer, ensure molecular weight markers were transferred; stain the membrane with Ponceau red, and the gel with Coomassie blue
f. Insufficient antigen binding to membrane 
If the antigen has low molecular mass, it may pass through the membrane. Switch to a membrane with a smaller pore size, or switch to a different type of membrane.
g. The antigen is masked by the blocking buffer 
Test different blocking buffers, such as milk, serum, BSA in Tris-buffered saline and phosphate-buffered saline. Test different concentrations of each.
h. Insufficient amount of antibodies present
Increase concentration of primary and/or secondary antibody.
i. Antibody exposure time is too short. 
Increase the exposure time.
j. Antibodies may have lost activity. 
Test antibodies by performing a Dot Blot.
k. Excessive washing of the membrane. 
Reduce the number of washes.
l. Substrate incubation is too short.
Increase substrate incubation time.
m. Substrate has lost activity 
Test substrate using a positive control.
n. Sodium azide is inhibiting enzyme reaction of HRP-conjugated antibody. 
Do not use sodium azide together with HRP-conjugated antibodies.

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