I have expressed my protein in E. coli. and I want to purify it. I performed the ammonium sulfate precipitation with 15%, 25%....75% in the indicial samples and ran both the supernatant and precipitates on SDS page. I noticed that my protein starts to precipitate even at 15% (at this conc half of the protein was in pellet and half was in supernatant). at 35% all the recombinant protein was precipitated and was present in gel. Now I want to do purification of that protein by Hydrophobic interaction chromatography using P-sepharose. I am wondering:
1. What should the concentration of ammonium sulfate in the sample be? (Normally it is recommended to be 1 to 1.5 M but at this my protein precipitates).
2. What should the conc of ammounium sulfate be for column equilibration?
3. What is the appropriate ammonium sulfate conc for wash buffer and for elusion buffer?
I think that you have answered the question yourself you cannot use HIC if you use ammonium sulfate. You need not use ammoniun sulfate for HIC you could use any high molarity salt such as NaCl. However, I suspect that you will have issues with precipitation with any high concentration salt.
Are you using an existing protocol? What protein are you purifying? knowing these this would help.
I have used HIC and it is fantastic for some proteins but for most applications the resolution is not very good. If your ammonium sulfate fraction is pure enough you may get away with SEC I use superdex 200 pg. Good luck
Hi Ted. Thanks for replying. Yeah I am using existing protocol first HIC and than q- column. Protein is amylase. With amm sulphate ppt all the junks come along with protein that r really difficult to separate afterward so can not use SEC even anion exchange did not work to separate these impurities.
Can you use a different salt other than ammonium sulfate for the HIC? Also there are many types of HIC columns I assume you are using pehnyl-sepharose you may want to try another.
The idea of using HIC and then ion exchange is sound because the reluctant for the HIC column is ready to load onto ion exchange without adjustment but this rarely works in practice.
This is just a though you may want to check your ammonium sulphate precipitation protocol many people do this wrong.
This website has a calculator for how much to add and at what temp
http://www.encorbio.com/protocols/AM-SO4.htm
good luck
hey thank u. i alway use this calculator. I am tryinh to switch to another method because it is not working.
If the anion exchange did not work have to tried a cation exchange column? Also did you remove the ammonium sulfate before running it on the ion exchange column? If it did bind, but you were unable to resolve the impurities you can try using a shallower elution gradient.
I did tried it too but now I have changed the strategy and I have recloned my protein with His tag. and purification is in process. Thank you all of you.
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