Positive EMSA result without the addition of a biotin-labelled probe

Question

[sofiablancoglez]

Hello!

I am asking this question because I have not found this problem described in any EMSA problems/limitations list.

I have loaded, in the 8 lanes you see in the figure below (the probes are always marked with biotin):

1. functional probe
2. non-functional probe
3. functional probe + mussel gonad protein extract
4. non-functional probe + mussel gonad protein extract
5. mussel gonad protein extract (without any DNA probe)
6. functional probe + trout liver protein extract
7. non-functional probe + trout liver protein extract
8. trout liver protein extract (without any DNA probe)

The 2 free probes (lanes 1 and 2) carry the same reagents as the rest of the samples except the protein extract (thus I rule out contamination of the binding buffer, DNA competitor, etc.) How can I get a retention band in lanes 5 and 8 where I have not loaded any labelled probes, but only the protein extract? Also, the retention band height I observe in trout and mussels is different (I rule out contamination of the native extraction buffer). Note that the bands below correspond to free probes.

Is there something in the protein sample that interacts with the luminol, and what changes in trout compared to mussels? I have seen that blood, for example, can interact with luminol giving a signal. Also, enzymes can be naturally bound to biotin (and maybe could produce the signal in the extract without adding a labelled probe), but is the biotin used in DNA tagging the same as the biotin found naturally in cells? I have also considered purifying the extract, but this could misfold the proteins. Why haven't I seen this problem described anywhere (from what I have read)? What mistake could I be making?

Thank you very much

[Mensur Dlakic]

What made you think that a website with a tagline "Bioinformatics explained" would be a good venue for your experimental problems?