1. Why are there multiple bands on my blot?

1. Why are there multiple bands on my blot?

Several variables may account for this:
a. An excessive amount of lysate loaded onto the gel may cause extra bands. Using decreasing dilutions of lysate will help to determine optimal loading amounts. An increased wash cycle may also alleviate this problem.
b. Lower molecular weight products may be due to protein degradation. We suggest preparing fresh samples by lysing cells/tissues according to our recommended protocol. Compare the signal of your experimental samples with the positive control.
c. Non-specific bands may be due to the secondary antibody. We recommend running a no primary antibody control: verify the appropriate dilution of the secondary detection system, run the Western blot with no primary antibody, and probe only with the secondary detection system.
d. Higher molecular weight products may be a result of post-translational protein modifications, including, but not limited to, glycosylation, myristylation, phosphorylation, and/or ubiquitination.
e. Additionally, higher molecular weight bands may also be a result of protein aggregation that is not resolvable by SDS and boiling.

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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?
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