Hello everybody,
Here is my question:
Does anybody know how to use ammonium acetate for protein hydrophobic interation cromatography (HIC)? What concentration should it be added to the protein sample? May ammonium acetate denature at such concentration?
I thank any help in advance.
One typically bgins with a high salt concentration, such as 1 M and elutes with a gradient of decreasing salt. Ammonium sulfate is commonly used, probably because it is gentle on proteins and has a higher ionic strength on a molar basis than ammonium acetate.
It all depends on the hydrophobicity of your protein, just try some high concentration, I usually start at 2 M (NH4)2SO4 (+ buffer), linear decreasing gradient of the salt.
If you worry about salting out effect, you can quickly test the initial conditions in a test tube.
If you wish to know more, i would suggest the handbook on HIC/RP from GE.
http://www.gelifesciences.com/vgn-ext-templating/v/index.jsp?storeId=GELifeSciences-us&vgnextoid=70563cc694b47310VgnVCM10000024dd1403RCRD
Add to your protein the buffer containing 1-2 M salt (buffer A) and load onto the HIC column, then perform the gradient with buffer B (same buffer but without salt). You can use NaCl or NaAcetate (usually 2M); NaCl and NaAcetate are more gentle salts than Ammonium sulphate (1M usually used). I hope it helps!
First perform a small-scale pilot experiment to determine at which concentration of ammonium acetate (or ammonium sulfate, which works better, and for which much more information is available) your protein starts to precipitate. For example: add the ammonium salt to 20, 30, 40............90 % saturation to 1 ml samples of your extract (the amounts of ammonium sulfate to be added to reach a certain saturation value can be read from tables, as presented in the Methods of Enzymology Protein Purification volume (volume 463), or you can use the calculator on this site: http://www.encorbio.com/protocols/AM-SO4.htm .This pilot experiment is quite important: some proteins tend to precipitate already at low saturation levels of (25 - 50 % saturation, or 1 - 2 M at 0 degree Celcius, (you will loose considerable amounts of your protein at these concentrations) others start to precipitate at much higher concentrations (e.g. 75 % saturation, or 3 M at 0 Celcius (these proteins hardly bind to the HIC columns at 1 M (25 %) ammonium sulfate. You should use a kosmotropic salt, like ammonium sulfate/acetate; Salts like NaCl or Na-acetate are not suitable for HIC; Ammonium sulfate is very gentle.
If your protein is an enzyme for which you have an assay, you can measure the activity of your enzyme in the supernatant to determine at what concentration your protein starts to precipitate. If your protein does not have enzymatic activity, you can try SDS PAGE (but: ammonium sulfate at high concentrations might disturb separation on SDS gels).
Having determined the concentration ammonium sulfate/acetate where your protein starts to precipitate, you add ammonium sulfate/acetate to a concentration slightly lower: i.e, your protein should just remain soluble. Remove precipitated proteins by centrifugation (16000 rpm, 10 min, in a 8 x 50 rotor in a High-Speed centrifuge). Equilibrate your HIC column (usually a column with covalently linked phenol groups, like Phenyl Sepharose) with the same concentration of ammonium sulfate/acetate in the buffer you use to keep your protein stable. Next.load your column with the clarified sample, and wash the column with your equilibration buffer to elute proteins that don't bind to the column. Last step is to elute your bound proteins, stepwise or with a gradient, with a gradient of decreasing concentration of buffered ammonium sulfate.
Is you don't detect your protein after the concentration of ammonium sulfate in your column decreased to 0 M, you can try a positive gradient (for example: 0 - 0.5 M) of ethylene glycol to elute your protein.
Ammonium sulphate is much better than ammonium acetate to perform hydrophobic chromatography. It's quite friendly even at high concentrations (around 2.5 M).
chromatographic gels such as Sepharose 4B can be used to carry out such as kind of purifications using linear gradients to elute the proteins.
Keep in mind that some proteins precipitate at high ammonium sulphate/acetate concentration so the first assay you have to run is related to the protein solubility in the presence of different ammonium sulphate/acetate concentrations.
Ammonium acetate is also an old-school pH buffer. It has little buffering capacity in the neutral area but people assumed that things would stay sufficiently neutral.
Make sure you use an appropriate pH buffer in your experiment of course. MOPS is excellent around pH 7 and cheap. Tris is terrible around pH 7 and cheap.
Dear all,
Thanks a lot for your very useful answer to my question. Some of you suggested the use of ammonium or sodium sulphate for HIC. Even though this is a valuable suggestion, I would not be able to use sulphate salts with my sample and that is because my question was specifically focused on ammonium acetate. My sample contains CHAPS as detergent (solubilized membrane proteins from mouse brain) and I have checked that mixture of these both CHAPS and sulphate salt induces precipitation of one of them (I do not know which one precipitates). In this sense, I have tested ammonium acetate compatibility with my detergent and it was right (no precipitation observed) and I have performed one experiment using sodium chloride for HIC and the result was not too good (as Prof. Walter Van Doogen mentioned above). In conclusion, ammonium acetate is the best option I consider right now to perform HIC from a membrane protein sample containing CHAPS as detergent.
Once again, I would like to thank all your help.
Regards
Manuel
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