I would keep the diluted aliquote at 4 degree C for a minimum time to achieve maximum functionality in experiments.

Dear Prof. Divaker Choubey,

I would like to know the time scales involved in degradation of Nucleosomes. Would it degrade in few days or weeks? And any estimation of degradation percentage would be helpful.

Hi, the answer to your question lies in how the nucleosomes (nucs) were prepared. You need that information. EpiCypher sells both recombinant (reconstituted) as well as cellular chromatin-derived nucs. The stability of both at 4C would highly depend on the contaminants and the methods of preparation. I have never used any commercial nucs; but I have extensively prepared and used both forms of nucs and will detail my opinion below.

If you got the HeLa/avian chromatin-derived nucs, you need to run an SDS-PAGE (of, say, 1ug nucs) and examine by simple staining the extent of impurity. If the four histone bands are in overwhelming majority, with negligible other bands, you may keep the nucs at 4C. But, I suggest you run about 100ng of nucs in a native 5% PAGE in either 1x Tris-glycine EDTA (no SDS) or in 0.5x TBE (both gels contains 5% glycerol) to see if the nucs are intact (slow migrating, band around 300bp) or have unraveled (additional band around 170-180bp). If you dont see this faster-migrating species over several days of storage at 4C, you are fine. Alternative storage could be at -20C AFTER you have added glycerol to final 50%, with additional 1mg/ml BSA (nuclease-protease free). Based on hi purity, HeLa or avian (from chicken liver chromatin) nucs in my experience can stay intact for many weeks at 4C and for years at -20C.

If you got the recombinant nucs, you need to know how it was reconstituted. You might already know that there are two most-preferred ways to reconstitute nucs. (1) using enzymes and chaperones (e.g., https://www.activemotif.com/catalog/13/chromatin-assembly-kit), and (2) using salt dilution/dialysis (e.g., https://www.neb.com/products/e5350-epimark-nucleosome-assembly-kit#Protocols%20&%20Manuals). The enzyme-chaperone method utilizes energy from ATP to form the nucs. These nucs are short-lived; you must use them within two days max. I have not used the NEB salt-dilution kit (link above), but if the histone purity is excellent, the salt-diluted nucs should stay intact for months at 4C.

I routinely reconstitute my own nucs and chromatin (on template DNA up to 8kb) using my self-made HeLa core histones (H1-depleted) and my salt dilution method. My nucs and chromatin stay intact for years at 4C.

So, here are the options for you: (1) speak to EpiCypher on how the nucs were made, their purity, and on storage conditions that they may have tried. Then, infer your options. (2) keep your nuc aliquots at 4C for different duration (say, 1d, 1week, 1month) and run about 100ng of nucs on a gel as suggested above. Remember: (1) you must thoroughly rinse the apparatus with distilled water if it was previously used for SDS-PAGE (so that you remove any trace of SDS); (2) you must stain the gel AFTER run with EtBr (do not add EtBr to the gel/running buffer); preferable if you stain the gel with non-toxic alternatives; (3) do NOT use the commercial 6x Loading Dye. They contain SDS and other components that will unravel nucs. Instead, add glycerol to the sample to final 10% and carefully load the samples into the gel looking at refraction (or, you may add a tiny amount of bromophenol blue so that the sample looks slightly bluish--just enough for you to load it). See for yourself how long your nucs stay intact at 4C.

Hope the above helps.