BCDX2 (15 μM) was incubated for 4 hours at 37°C with and without chymotrypsin (1.5 μM) in HGMT buffer containing 150 mM NaCl, and 0.5 mM ADP.BeFx (0.5 mM ADP, 0.5 mM BeSO4 and 10 mM NaF). The samples were then gel filtered using a Superdex 200 Increase 3.2/300 GL column (Cytiva) equilibrated in HMT buffer (25 mM HEPES pH 7.5, 2.5 mM MgCl2, 0.25 mM TCEP) with 100 mM NaCl on a Micro-kit equipped ÄKTA pure. Fractions were taken and analysed on 5 SDS-PAGE gels. One was directly visualised with Quick Coomassie stain (Generon) and visualised on a ChemiDoc MP Imaging system. The others were analysed by western blotting with antibodies against RAD51B (Rabbit polyclonal, 1:1000 SWE32, this lab), RAD51C (Rabbit polyclonal, 1:1000, SWE6856 (link)), RAD51D (Rabbit monoclonal, 1:1000, Abcam ab202063) and XRCC2 (Rabbit polyclonal, 1:1000, SWE3556 (link)). Membranes were incubated with Alexa Fluor Plus 800 anti-rabbit secondary antibody (1:40,000, Invitrogen A32735) and imaged using an Odyssey DLx instrument with ImageStudio software (Licor).
Superdex 200 increase 3.2 300 gl column
Manufactured by Cytiva
The Superdex 200 Increase 3.2/300 GL column is a size exclusion chromatography column designed for protein and biomolecule separation and purification. It features a high-performance resin with a broad separation range for molecules between 10 and 600 kDa. The column is compatible with a variety of buffers and solvents, making it suitable for use in diverse applications.
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2 protocols using superdex 200 increase 3.2 300 gl column
1
Chymotrypsin-Mediated RAD51 Complex Analysis
2
Chymotrypsin-Mediated RAD51 Complex Analysis
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BCDX2 (15 μM) was incubated for 4 hours at 37°C with and without chymotrypsin (1.5 μM) in HGMT buffer containing 150 mM NaCl, and 0.5 mM ADP.BeFx (0.5 mM ADP, 0.5 mM BeSO4 and 10 mM NaF). The samples were then gel filtered using a Superdex 200 Increase 3.2/300 GL column (Cytiva) equilibrated in HMT buffer (25 mM HEPES pH 7.5, 2.5 mM MgCl2, 0.25 mM TCEP) with 100 mM NaCl on a Micro-kit equipped ÄKTA pure. Fractions were taken and analysed on 5 SDS-PAGE gels. One was directly visualised with Quick Coomassie stain (Generon) and visualised on a ChemiDoc MP Imaging system. The others were analysed by western blotting with antibodies against RAD51B (Rabbit polyclonal, 1:1000 SWE32, this lab), RAD51C (Rabbit polyclonal, 1:1000, SWE6856 (link)), RAD51D (Rabbit monoclonal, 1:1000, Abcam ab202063) and XRCC2 (Rabbit polyclonal, 1:1000, SWE3556 (link)). Membranes were incubated with Alexa Fluor Plus 800 anti-rabbit secondary antibody (1:40,000, Invitrogen A32735) and imaged using an Odyssey DLx instrument with ImageStudio software (Licor).
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