I’m doing a northern blot assay to detect transfected siRNAs on cell culture lysates. Several studies describes that liquid Northern hybridization is a more sensible method to my intended assay. However, most protocols uses isotopes marked probes that are not available in my lab.
My plan is to hybridize my sample with my biotinylated-probe, treat it with Exonuclease I, and then load this on a non-denaturing 15% polyacrylamide gel. After this I would treat the gel with a streptavidin-HRP solution and ECL solution. My questions are:
1- Is that possible that the streptavidin “enter” the gel and ligate to biotin markers on the probe?
2- If the streptavidin ligate to the biotin, will the HRP on it still able to activate ECL?
There are a few reports of detection of in-gel detection of biotinylated proteins but, is it possible to do it with biotin-probes?
See this paper:
Nature Protocols 3, - 1077 - 1084 (2008)
Published online: 5 June 2008 | doi:10.1038/nprot.2008.67
Seems like you'd want to transfer to a membrane, similar to a western blot, before detection.
Hi Bruno! Did you ever end up doing that experiment? Interested in knowing if it worked...
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