This antibody has been shown by an independent laboratory to work in RNA-IP-seq. Please use at an assay-dependent dilution.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Note: This protocol is written for spotting either purified
total RNA or poly A-purified mRNA (titration of 2 μg, 1 μg, 500 ng,
250 ng, 125 ng, 62.5 ng, and 31.25 ng) onto a positively charged nylon
membrane using a 96-well dot blotting apparatus. Depending on the source of
the RNA, more or less RNA may be required for detection with the antibody.
• RNA is sensitive to degradation by RNases, which can affect sample integrity. It is recommended that all surfaces and equipment undergo RNase decontamination.
• Purify total RNA and/or mRNA from cell pellet using an RNA isolation kit. Assess total RNA quality by gel electrophoresis on a 1% agarose gel. The 28S and 18S RNA should present as distinct bands. Smearing indicates RNA degradation. See Figure 1.
• Cut a piece of nylon membrane to fit the size of the dot blot manifold.
• Wet nylon membrane with 10X SSC Buffer.
• Dry membrane by placing it in a 96-well dot blot apparatus and applying vacuum.
Optional: To normalize sample loading using methylene blue,
apply stain before Section C, Step 1 and capture an image. Rinse blots three
times for 5 min each with 15 mL dH2O. Stain does not affect
antibody binding or detection.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr.
Figure 1. Representative image of isolated, intact total RNA. 28S and 18S RNA should migrate as distinct bands. If RNA presents as a smear, the sample may be degraded and unfit to use in downstream assays. Lane 1 is NEB 100 bp DNA ladder and lane 2 is total RNA isolated from 293T cells.
posted November 2018
Protocol Id: 1784
All Species Expected
Monoclonal antibody is produced by immunizing animals with N6-methyladenosine.
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