Nytran supercharge nylon membrane, by GE Healthcare - Product details

1

Mitochondrial RNA Capping Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

NCIN capping of DNAzyme-generated subfragments of mitochondrial RNA were analyzed by a procedure consisting of: (i) electrophoresis on 7.5 M urea, 1x TBE, 10% polyacrylamide gels supplemented with 0.2% 3-acrylamidophenylboronic acid (Boron Molecular); (ii) transfer of nucleic acids to a Nytran supercharge nylon membrane (GE Healthcare Life Sciences) using a semidry transfer apparatus (Bio-Rad); (iii) immobilization of nucleic acids by UV crosslinking; (iv) incubation with a ³²P-labelled oligodeoxyribonucleotide probe complementary to the 5'-end containing subfragments of target RNAs (JB555, COX2 RNA; JB525, 21S RNA; JB515, LSP-derived RNA; ³²P-labelled using T4 polynucleotide kinase and [γ³²P]-ATP [Perkin Elmer]); (v) high-stringency washing, procedures as in (Goldman et al., 2015 (link)); and (vi) storage-phosphor imaging (Typhoon 9400 variable-mode imager; GE Life Science).
Bands corresponding to uncapped and NCIN-capped DNAzyme-generated subfragments were quantified using ImageQuant software. The percentages of uncapped RNA (5'-ppp), NAD⁺-capped RNA (5'-NAD⁺), or NADH-capped RNA (5'-NADH) to total RNA were determined from three biological replicates.

Bird J.G., Basu U., Kuster D., Ramachandran A., Grudzien-Nogalska E., Towheed A., Wallace D.C., Kiledjian M., Temiakov D., Patel S.S., Ebright R.H, & Nickels B.E. (2018). Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase. eLife, 7, e42179.

+ Open protocol

+ Expand

2

RNA Detection and Quantification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

In Figure 3b–c, RNA products were detected by a procedure comprising electrophoresis on 10% 7.5 M urea slab gels (equilibrated and run in 1× TBE), transfer to and UV cross-linking to a membrane (Nytran supercharge nylon membrane, GE Healthcare Life Sciences), hybridization with a ³²P-labeled “locked-nucleic-acid” probe complementary to positions +31 to +42 of RNA-standard (Sequence, agCaaAttAacCc; LNA bases capitalized; purchased from Exiquon, Woburn, MA; ³²P-labeled using T4 polynucleotide kinase), high stringency washing (procedure as described in²⁵), and storage-phosphor imaging. Bands were quantified using ImageQuant software. The % of RNA products in the upper band was determined for each sample as: 100 * (upper band/(upper band + lower band)). Values of %NudC-sensitive products represent the reduction in the % of RNA products in the upper band upon treatment with NudC. Values shown represent the average of three technical replicates (in vitro) or three biological replicates (in vivo).
For the experiments in Figure 3c, values of half-life were determined by fitting data to a single-exponential decay function (mean±SEM of three biological replicates for exponential phase and five biological replicates for stationary phase).

Bird J.G., Zhang Y., Tian Y., Panova N., Barvík I., Greene L., Liu M., Buckley B., Krásný L., Lee J.K., Kaplan C.D., Ebright R.H, & Nickels B.E. (2016). The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA. Nature, 535(7612), 444-447.

+ Open protocol

+ Expand

3

RNA Detection and Quantification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

In Figure 3b–c, RNA products were detected by a procedure comprising electrophoresis on 10% 7.5 M urea slab gels (equilibrated and run in 1× TBE), transfer to and UV cross-linking to a membrane (Nytran supercharge nylon membrane, GE Healthcare Life Sciences), hybridization with a ³²P-labeled “locked-nucleic-acid” probe complementary to positions +31 to +42 of RNA-standard (Sequence, agCaaAttAacCc; LNA bases capitalized; purchased from Exiquon, Woburn, MA; ³²P-labeled using T4 polynucleotide kinase), high stringency washing (procedure as described in²⁵), and storage-phosphor imaging. Bands were quantified using ImageQuant software. The % of RNA products in the upper band was determined for each sample as: 100 * (upper band/(upper band + lower band)). Values of %NudC-sensitive products represent the reduction in the % of RNA products in the upper band upon treatment with NudC. Values shown represent the average of three technical replicates (in vitro) or three biological replicates (in vivo).
For the experiments in Figure 3c, values of half-life were determined by fitting data to a single-exponential decay function (mean±SEM of three biological replicates for exponential phase and five biological replicates for stationary phase).

Bird J.G., Zhang Y., Tian Y., Panova N., Barvík I., Greene L., Liu M., Buckley B., Krásný L., Lee J.K., Kaplan C.D., Ebright R.H, & Nickels B.E. (2016). The mechanism of RNA 5′ capping with NAD+, NADH, and desphospho-CoA. Nature, 535(7612), 444-447.

+ Open protocol

+ Expand

Nytran supercharge nylon membrane

Lab products found in correlation

Close spelling variants of this product

3 protocols using nytran supercharge nylon membrane

Mitochondrial RNA Capping Analysis

RNA Detection and Quantification Protocol

RNA Detection and Quantification Protocol

About PubCompare

Ready to get started?