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G 25 sephadex columns

Manufactured by GE Healthcare
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The G-25 Sephadex columns are size exclusion chromatography columns used for the separation and purification of proteins, peptides, and other biomolecules. These columns utilize the Sephadex G-25 resin, which is a cross-linked dextran-based material, to facilitate the separation of molecules based on their size and molecular weight. The G-25 Sephadex columns are commonly used in various applications, such as buffer exchange, desalting, and sample cleanup.

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Lab products found in correlation

T4 polynucleotide kinase, by New England Biolabs (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Hybond nylon membrane, by GE Healthcare (1 mentions) Dig high primer dna labeling and detection starter kit 2, by Roche (1 mentions) Dig detection kit, by Roche (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Ultrahyb oligo buffer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Sephadex G-25 column PD10 Sephadex G-25 desalting columns Sephadex-G-25 PD-10 pre-packed columns PD-10 columns (Sephadex G-25) PD-10 Columns SephadexTM G-25 M HiPrep Sephadex G-25 Desalting columns Sephadex G-25 PD10 columns NAP-5 Sephadex G-25 DNA Grade columns PD‐10 desalting columns containing Sephadex G‐25 resin PD MiniTrap™ Sephadex G-25 columns

4 protocols using g 25 sephadex columns

1

Radiolabeling and Hybridization of Oligonucleotides

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Oligonucleotide sequences for all substrates are provided in Table 1. For all but one radiolabeled substrate, 10 pmol of the ‘A’ oligonucleotide was 5’ labeled using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and γ−32P-ATP (Perkin Elmer, Pittsburgh, PA) at 37°C for 1 hr. In the case of Figure 2D, oligonucleotide B was labeled instead of A, but other conditions were the same as described. After labeling reaction, 15 pmol of oligonucleotides B (and C when appropriate) were added in a solution of 40 mM Tris-HCl, pH 8.0 and 50 mM NaCl. Hybridization reactions were placed in a heat block at 85°C and allowed to cool naturally to room temperature. Unlabeled substrates were prepared exactly as above, but without the γ−32P-ATP and T4 polynucleotide kinase. Radiolabeled probes were applied to G25 sephadex columns (GE Life Sciences, Pittsburgh, PA) to remove unbound nucleotides and then analyzed by non-denaturing polyacrylamide gel electrophoresis for complete hybridization.
Baptiste B.A., Baringer S.L., Kulikowicz T., Sommers J.A., Croteau D.L., Brosh RM J.r, & Bohr V.A. (2021). DNA Polymerase β Outperforms DNA Polymerase γ in Key Mitochondrial Base Excision Repair Activities. DNA repair, 99, 103050.
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2

Northern Blot Analysis of GFP mRNA

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RNA (‘northern’) blot analysis of GFP mRNA was performed as described previously (Du et al., 2014a (link)). Total RNA was extracted from agroinfiltrated leaves using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. Five micrograms of each total RNA sample was separated on 1.5% agarose gel containing 7% formaldehyde, and transferred onto Hybond+nylon membrane (GE). GFP mRNA was detected by the digoxigenin (DIG)-labeled DNA probes that were prepared using the DIG high primer DNA labeling and detection starter kit II (Roche) as recommended by the manufacturer, and cleaned up using G25 Sephadex columns (GE). DIG-labeled probes were detected using a chemiluminescence-based DIG detection kit (Roche) according to the manufacturer’s instructions.
Gao Y., Yang J., Zhang X., Chen A., Gu Z, & Du Z. (2021). The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression. Frontiers in Microbiology, 12, 760937.
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3

Detection of tRNA-derived Small RNAs

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For detection of tsRNAs, 30 μg of total RNA in 2× Novex TBE–urea sample buffer (2×) (Thermo Fisher Scientific) and separated on 12% denaturing (50% g/v urea) polyacrylamide gel in 1× TBE. RNA was transferred onto hybond-N + nylon membrane at 5 V for 1 h followed by UV-crosslinking and pre-hybridization in ULTRAhyb-Oligo buffer (Thermo Fisher Scientific) at 42°C for at least 30 min. tRNA/tsRNA-specific oligonucleotide probes were radiolabelled with 32P-ATP by poly nucleotide kinase (PNK) (PerkinElmer) at 37°C for 1 h. Radio-labelled probes were purified in G-25 Sephadex columns (GE Healthcare) and hybridized onto the membrane at 42°C followed by washes with 0.1× SCC washing buffer (0.05% SDS) and subjected to autoradiography.
Di Fazio A., Schlackow M., Pong S.K., Alagia A, & Gullerova M. (2022). Dicer dependent tRNA derived small RNAs promote nascent RNA silencing. Nucleic Acids Research, 50(3), 1734-1752.
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4

EMSA Analysis of vtRNA1-1 Regulatory Regions

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For EMSA the vtRNA1-1 DNA locus including its upstream and downstream regulatory regions was amplified with PCR (primers Supplementary Table 2). The amplified 438 bp long PCR product was gel purified and 5′-[32P] end-labeled. Unincorporated ATP was removed with G-25 Sephadex columns via centrifugation (GE Healthcare). 5 μg of nuclear protein extract was incubated with Stabilization Solution D2 as well as Binding Buffer B2 (both active motif) for 20 min at 4°C. For super-shift experiments, nuclear proteins were incubated with human NFκB p65 antibodies (Santa Cruz, sc-372 X) for 30 min at 4°C. 25,000 cpm of radiolabeled probe together with Binding Buffer C2 as well as Stabilization Solution (active motif) were incubated with the reaction mixture for 20 min at 4°C. For competition a 50–fold excess of unlabeled PCR product was added 10 min before the addition of the radiolabeled probe. The samples were separated on a 5% native polyacrylamide gel for 3.5 hours, vacuum-dried and afterwards exposed on a phosphoimager screen overnight.
Amort M., Nachbauer B., Tuzlak S., Kieser A., Schepers A., Villunger A, & Polacek N. (2015). Expression of the vault RNA protects cells from undergoing apoptosis. Nature Communications, 6, 7030.
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