Hello,
I am posting this question because I have not found this issue described in any EMSA problems/limitations list. My main objective is to detect the retention (electrophoretic mobility shift) of a biotin-labelled DNA probe (which will be revealed with luminol) due to its interaction with a factor theoretically present in a protein extract. This would be observed as a retained band (shift band) at the top of the gel if there is an interaction, while the remaining free DNA probe would appear at the bottom of the gel.
In the attached photograph of the nylon membrane, the following sample types can be visualised (the probes are always marked with biotin):
1. Functional DNA probe
2. Non-functional DNA probe
3. Functional DNA probe + protein extract organism 1
4. Non-functional DNA probe + protein extract organism 1
5. Protein extract organism 1 (without any DNA probe)
6. Functional DNA probe + protein extract organism 2
7. Non-functional DNA probe + protein extract organism 2
8. Protein extract organism 2 (without any DNA probe)
The 2 free DNA 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, or any reagent involved in the hybridisation reaction). How can I get a retention band in lanes 5 and 8 where I have not loaded any labelled DNA probes, but only the protein extract? Also, the retention band I observe in both organisms is different (thus I rule out contamination in the native extraction buffer). Note that the bands below correspond to free probes.
Could there be something in the protein extract sample that interacts with the luminol? I have seen that blood, for example, can interact with luminol producing a signal. Also, enzymes can be naturally bound to biotin, 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, isn’t it? Why haven't I seen this problem described anywhere (from what I have read)? What mistake could I be making?
Thank you very much
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