Hi Josy,

For reconstituting anaerobically, just apo-protein, Fe salts and Na2S are more than sufficient. However, if you are trying to purify holo-protein (with FeS clusters), then you may have to co-express other plasmids containing either Isc or suf operons.

Bhanu P Jagilinki Thank you for your answer, do you have experience with reconstituting Fe-S clusters? If so then maybe you can give me some advice! Usually I purify the protein with the Fe-S cluster (without the need to co-express Fe-S producing proteins), but now I'm actively trying to loose the cluster to observe what happens and then the idea is to reconstitute the Fe-S again and observe if activity is restored. However I do not have an anearobic glove box on site. I do have argon which I regularly use to flush buffers. I also have Ammonium iron(II) sulfate and sodium sulfide, but I read that IscS or Nsp1 often provide a better sulfide-source as it is gradually & freshly made (and therefore less oxidation). Do you think reconstitution will work if I solubilise the salts in argon flushed buffer/water? If an anaerobic glove box is required, please let me know, then I'll try to arrange that I can use one. And do you then let the Fe-S cluster form first before adding it to the apo-protein or do you directly add the Fe and Na2S salts to the protein?

Among FeS proteins [4Fe-4S] are highly o2 sensitive, and their half lives (t1/2) are around 30 to 70 minutes. So, for [4Fe-4S] proteins, anaerobic glove chamber is a must.

Argon flushed buffers are good, but for very short time. The protein clusters oxidize upon exposure to air, without anaerobic chamber.

I advise you, move over to iron (iii) salts like FeNO3, or Fecl3 or Ferric ammonium citrate.

It does not matter from where the S source is coming form, as long as the holo-protein, is not inside the anaerobic chamber or if it is not properly air tight sealed inside the anaerobic chamber, you will loose the clusters.

So, for all these problems, the anaerobic chamber is the only solution.

For the last question, Always apo-protein has to be taken first, reduce with DTT, then add Fe salts, then lastly add Na2s. If you mix fe and Na2S in buffer, without apo-protein, you will tend to form several unwanted inorganic complexes.

Good luck

Bhanu