Consensus sequences for human histones H2A, H2B and H3.2 and Xenopus histone H4 were cloned into pST50 expression vectors, expressed in BL21(DE3)pLysS Escherichia coli, purified from inclusion bodies and reconstituted into H2A–H2B dimers or H3–H4 tetramers exactly as previously described (58 (link)). Histone H3 was purified from inclusion bodies, solubilized in 7 M guanidine and purified by size exclusion chromatography (Sephacryl S300, GE Healthcare) followed by Source S cation exchange chromatography as previously reported (59 (link)). After purification, the histone was lyophilized and resuspended in water with 1 mM DTT. Plasmids containing repeats of widom 601 sequences (60 (link)) were gifts from Song Tan (61 (link)). Plasmids were amplified in HB101 E. coli and purified by alkaline lysis. Nucleosomal DNA fragments were excised from plasmid DNA using EcoRV and purified by anion exchange chromatography with a Source Q resin (GE Healthcare). Nucleosomes and tetrasomes were prepared by salt gradient dialysis as previously described (45 (link),58 (link),59 (link)). FLAG-tagged histone H2A–H2B dimers were prepared by coexpression and PEI precipitation followed by cation exchange chromatography using a Source S resin (GE Healthcare) as previously described (58 (link)).
Source s resin
Manufactured by GE Healthcare
Source S resin is a cation exchange chromatography media designed for the purification of biomolecules. It features a sulfopropyl functional group that facilitates the capture and separation of positively charged molecules. The resin is available in various bead sizes and can be used in a range of chromatographic applications.
Automatically generated - may contain errors
2 protocols using source s resin
1
Recombinant Histone Reconstitution and Purification
2
Purification of Mouse and Human cGAS Catalytic Domains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mouse cGAS catalytic domain containing residues 141–507 (mcGAS) was prepared as previously described4 (link). The human cGAS catalytic domain (residues 157–522) was prepared as previously described44 (link). The 6×His-Sumo tagged mouse cGAS catalytic domain (6×His-Sumo-mcGAS) used in the TR-FRET assay was expressed in BL21(DE3)pLysS cells. Cells were grown in 2XTY medium at 37 °C, and protein expression was induced through the addition of 0.2 mM IPTG when the OD600 reached 1.2 for 18 h at 16 °C. The 6×His-Sumo-mcGAS protein was purified using metal-affinity chromatography with Talon resin (Clontech, 635504), ion-exchange chromatography with Source S resin (GE Healthcare, 17094405) and size-exclusion chromatography with a Superdex 75 increase 10/300 GL column (GE healthcare), concentrated, supplemented with 20% glycerol and stored at −80 °C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!