Biotinylated RNA on Nylon Membrane - (Jul/08/2013 )
I'm in-vitro transcribing RNA biotin labelled and I'm running it in PAGE-Urea gel for subsequent blotting on Nylon membrane and streptavidin detection. I'm trying to set the staining protocol of the biotinylated RNA bound on the Nylon membrane with streptaviding conjugated fluorophore (IRDye 800CW). Right now I'm saturating the nylon membrane with the blocking buffer Odyssey from Licor (used also for WB) with 1% SDS and I'm performing the streptavidin incubation in the same solution for one hour, then I perform washes 5 min each with TBS-Tween 0.01% and SDS1%.
I easily end up with very high background and not clear bands.
Does someone has experience in streptavidin staining on nylon membrane? which is the best protocol biotin-streptavidin detection on nylon membrane?
No experience with streptavidin/biotin on nylon, but SDS may be too high and causing the block to not bind efficiently. The odyssey buffer is supposed to be good for this sort of assay, so it probably isn't that.
Have you tried a titration of the streptavidin based bit? If not, these are commonly used at 1:10,000 or higher dilution..
I'm indeed wondering about the high concentration of SDS, I might use 0.1% instead 1%, and evaluate is using TBS 0.1% SDS plus tween 0.1% in the washing steps. About the Streptavidin concentration I didn't try a titration, I've always used 1:5000 dilution and I've just increased the incubation time from 30 min to 1h which works much better.
Thanks for the reply!