Oligos corresponding to the GATA-like element, MBS1 and MBS2 of the Vdac2 promoter were annealed into double strands. Their sequences are described in Table S1 . EMSA was performed as described previously.54 (link) Briefly, the annealed oligos (250 ng) were incubated with 20 μCi [γ−32P] dATP and T4 polynucleotide kinase (Promega, M180A) for 1 h at 37°C to generate the radiolabeled probes, which were subsequently used in a G-50 column (Amersham Biosciences, 27-5330-01) to separate from free nucleotides for purification.
G 50 column
Manufactured by Cytiva
The G-50 column is a gel filtration chromatography column designed for the separation and purification of macromolecules. It is used to separate molecules based on their size and molecular weight. The column is packed with a porous gel material that allows smaller molecules to enter the pores, while larger molecules are excluded, resulting in their faster elution.
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Lab products found in correlation
2 protocols using g 50 column
1
EMSA Analysis of VDAC2 Promoter
2
Murine Angptl2 Antisense Probe Synthesis
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pBlueScript SK ( + ) vector (Addgene #212205) containing the target DNA sequence for murine Angptl2—subcloned using EcoRI/HindIII restriction sites between the T3 and T7 promoters—was linearized with the restriction enzyme MluI (R3198S, New England Biolabs) and purified through silica membrane spin columns (20021, Qiagen). The synthesis of a 980 bp anti-sense probe was then realized with 500 ng of linearized DNA incubated with DIG labelling mix (11277073910, Sigma) and T3 polymerase (M0378S, New England Biolabs). The transcription product was further treated with DNase (74204, Qiagen) to clear the probe from all residual DNA. The RNA probe was then purified through a G-50 column (CA95017-623L, Amersham) and stored at −30 °C.
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