We followed the method described previously [20 (link)]. We used Dynabeads M-280 Streptavidin-coated magnetic beads (Invitrogen, Waltham, MA, USA) for the in vitro pull-down analysis, which can be used to trap biotinylated molecules for multiple purposes [76 (link),77 (link),78 (link)]. We used them to capture biotinylated DNA, and checked whether AS2D-FLAG and as2 variant D-FLAG could bind to this DNA. We used nonbiotinylated oligos to ensure that the DNA did not bind directly to the beads in detectable amounts. The immunoblotting for FLAG-tagged proteins was performed using an anti-FLAG antibody (Wako, Osaka, Japan) and an anti-mouse goat antibody (Cosmo Bio Co. Ltd., Tokyo, Japan). The signal was detected using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Dynabeads m 280 streptavidin coated magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads M-280 Streptavidin-coated magnetic beads are uniform, superparamagnetic polymer beads with a diameter of 2.8 μm. The beads are coated with streptavidin, a high-affinity biotin-binding protein. These beads are designed for the isolation and purification of biotinylated molecules, including proteins, nucleic acids, and cells.
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Lab products found in correlation
2 protocols using dynabeads m 280 streptavidin coated magnetic beads
1
In vitro pull-down analysis of DNA-binding
2
Enrichment of Photoreactive Peptides
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A volume of tryptic digest corresponding to 80 pmol of photo(R/W) 9 CPP was incubated with 200 µg of streptavidin magnetic beads (Dynabeads M280 streptavidin coated magnetic beads from Invitrogen, Carlsbad, New Mexico, USA) for 60 min at RT under gentle stirring. Beads were conditioned before use and washed after incubation [19] . Peptides were eluted from the beads with 5 µL of 0.1 M hydrochloric acid (HCl) under gentle stirring (30 min).
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