10. Nonspecific Bands

10. Nonspecific Bands

Possible Cause & Solution
a. Non-specific antibody binding. 
Reduce primary antibody concentration
Decrease the amount of total protein loaded on gel
Adjust membrane blocking conditions
Increase number of washes
Verify the specificity of the antibody
Blot with the secondary antibody alone. If bands develop, choose an alternate secondary antibody
b. Degradation of protein. 
Prepare fresh samples. Use protease inhibitors during sample preparation. Minimize freeze/thaw cycles of sample.
c. Aggregation of analyte.
Increase the amount of DTT (20 -100mM) to ensure complete reduction of disulfide bonds. Heat in boiling water bath for 5-10 minutes before loading onto gel.
Cell lines that have been passaged many times may accumulate differences in their protein expression profiles. Go back to the original non-passaged cell line and run the current and original cell line samples side-by-side.
The protein sample has multiple modifications in vivo such as acetylation, methylation, glycosylation, phosphorylation, etc. Review the literature for modified protein variants. Adjust sample preparation accordingly.
Target protein has multiple isoforms, or other proteins share similar epitopes. Check the literature for target protein isoforms. Perform a BLAST search to check for possible cross-reactions. Include other cell or tissue types.

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