Can someone shared a protocol for immuno nothern blot (I am detecting biotin labelled tRNA derived fragment and detecting with an antibody, not with the probe). I tried according to Mishima E, Abe T. Immuno-Northern Blotting: Detection of Modified RNA Using Gel Separation and Antibodies to Modified Nucleosides. Methods Mol Biol. 2019;1870:179-187. doi: 10.1007/978-1-4939-8808-2_13. PMID: 30539555. But it is giving high background for me. Tried several ways of optimization, but I could not see a significant improvement.
You're using a combination of techniques:
Northern blot used to detect specific RNA molecules in a sample.
Biotin labeling*: Biotin is attached to the tRNA-derived fragment, allowing for detection.
Immunodetection is used to detect the biotin label.
This approach is often referred to as a "streptavidin-based detection" or "biotin-streptavidin detection" rather than "immuno Northren
To detect biotin-labeled tRNA fragments, you would typically use:
1. *Streptavidin conjugated to an enzyme* (e.g., HRP or AP) or a fluorescent tag.
2. *Alternatively*, an anti-biotin antibody can be used, followed by a secondary antibody conjugated to an enzyme or fluorescent tag.
The detection method would involve:
1. *Separation of RNA fragments* by electrophoresis (Northern blot).
2. *Transfer of RNA fragments* to a membrane.
3. *Detection of biotin-labeled fragments* using streptavidin or anti-biotin antibody.
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