Sample Prep & Detection Kits
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Accessory reagents & controlsBiochemicals
BiochemicalsProteins and Peptides
Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
Low endotoxin, Azide free.
Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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Reagents
Equipment
Dilute the RNA samples or oligos containing the modification of interest to an appropriate concentration with RNase free water.
Denature the diluted RNA sample at 95 degrees in a heat block to disrupt secondary structures for 3 min.
Chill the tubes on ice immediately after denaturation to prevent the re-formation of secondary structures of RNA.
Cut the positively-charged membrane to an appropriate size.
Mix the RNA sample by pipetting up and down and drop 1 µL of RNA onto the positively charged nylon membrane.
Let the pipetted RNA droplet diffuse onto the membrane via surface tension. Change tips after each loading, even between the same sample.
Transfer the dish with the membrane immediately into the chamber of SG Linker.
Cut the membrane according to the grid and transfer to a clean 24 well plate.
Wash the membrane in 500 μl of wash buffer for 5 min at room temperature with gentle shaking to wash off the unbound RNA.
Incubate the membrane (RNA side down) in 300 μl blocking buffer for 1 h at room temperature with gentle shaking.
Wash the membrane in wash buffer for 10 min.
Dilute the nucleoside competitors serially in blocking buffer to give final concentrations of 1000/200/40/8/0 nM.
Incubate the membrane with antibody in 300 μl overnight at 4 degrees C with gentle shaking.
Wash the membrane (flip the membrane so the RNA side faces up) three times for 10 min each in 10 ml of wash buffer with shaking.
Incubate the membrane (RNA side down) with an appropriate secondary IgG-HRP antibody diluted in blocking buffer for 1 h at room temperature with gentle shaking.
Wash the membrane (RNA side up) four times for 10 min each in 10 ml of wash buffer with shaking.
Incubate the membrane with ECL substrate (eg ab133406) for 5 min.
Expose the membrane with your imaging system and take images on high-resolution mode from 1 s exposure up to 3 min exposure with certain intervals.
If oligos are biotinylated, probe with a Streptavidin-AlexaFluor as a loading control. Ensure the ECL from the previous imaging has been quenched; thorough washing will aid this.
Wash the membrane in wash buffer for 30 min.
After fluorescence imaging, transfer the membrane to a petri dish containing 10 ml Methylene blue staining buffer.
For loading control of non-biotinylated RNA either methylene blue or ethidium bromide can be used.
Wash the membrane with tap water until the background is clean (around 30 s to 60 s wash).
Take a photo of the methylene blue stained membrane with your imaging system.