To ensure that pBpa-Hop (Extended Data Fig. 8a , b , d ) and pBpa-GR (Extended Data Fig. 10 ) cross-link the bound segment in the context of the loading complex, cross-linking reactions were performed immediately after the complex was fractionated from SEC (see the previous section). Using a UV-transparent, 96-well microplate (Corning) as a fraction collector, the whole fractions of the eluted GR-loading complex were subjected to UV exposure using an agarose gel imaging system (Enduro GDS Imaging System). Samples were irradiated for 60 min in total. To prevent overheating, the 96-well plate was placed on a shallow, UV-transparent plate filled with constantly refreshed ice water during the time course of the exposure. SDS–PAGE gels were used to analyse cross-linked products, followed by western blot transfer to nitrocellulose and probed with anti-MBP (New England BioLabs), anti-Hsp70 (Enzo Life Sciences) or anti-STIP1 (Proteintech) antibodies.
Enduro gds imaging system
Manufactured by Corning
The Enduro GDS Imaging System is a laboratory equipment product designed for imaging and analysis. It serves as a core function for tasks involving sample imaging and data collection.
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2 protocols using enduro gds imaging system
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Cross-linking of GR Loading Complex
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Crosslinking of GR-loading Complex
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To ensure that Hop crosslinks the bound segment in the context of the loading complex, crosslinking reactions were performed immediately after the complex was fractionated from SEC (see the above complex preparation section). Using a UV-transparent, 96well microplate (Corning) as a fraction collector, the whole fractions of the eluted GRloading complex were subjected to UV exposure using an agarose gel imaging system (Enduro GDS Imaging System). Samples were irradiated for 60 mins in total. To prevent overheating, the 96-well plate was placed on a shallow plate filled with constantly refreshed ice water during the time course of the exposure. SDS-PAGE was used to analyze cross-linked product, followed by Western plot transfer to nitrocellulose and probed with an MBP antibody (New England BioLabs) (Extended Data Fig. 27).
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