I am looking at the proteome of soils with a high humic content, however the analysis is always complicated by humic acids, which co-extract with almost every purification procedure. Therefore I am going to try to remove the bulk of the humic acids with XAD 4 ion-exchange resin and XAD 8. I was wondering if anybody had any experience with this and what was the best way to construct the columns?
If you want to remove the humic acid, you can do an extract in 0.1 M NaOH (or KOH). A good ratio is 2.5 g of material to IL of 0.1 M HNaOH. Then centrifuge the extract to remove nonsolubles. The supernatant is then pooled and acidified to pH 1 with conc. HCl. This will cause the HA to precipitiate. The HA can then be removed by centrifugation. The supernatant contains the FA and other base and acid soluble components. The FA can be adsorbed to DAX-8 resin. If you us DAX-8 and XAD-4 in tandem (DAX-8 has to be used first because the FA molecules are to big to flow through the small pores of the XAD-4 resin). The XAD-4 resin will adsorb "XAD-4 hydrophilic acids". Lower molecular weight carboxylic acids are in the eluate.
Hi Geryy,
did you already tried to remove the bulk of the humic acids with XAD 4 ion-exchange resin and XAD 8? How did it work?
Thank you
Peter
Use of XAD 8 and XAD 4 for Humic and Fulvic acid recovery from dissolved organic carbon
https://pubs.er.usgs.gov/publication/70016824
My solution was not to let the humic acids come into solution in the first place, hence creating a cleaner extract
Here is another method I found recently
Article A simple cleanup method for the removal of humic substances ...
Gerry,
The use of XAD-8 (now DAX-8) and XAD-4 was developed by the US Geological Survey in Boulder, CO to fractionate DOC. If you want to use this technique you will first have to extracted the humic substances (HA/FA) in e.g. 0.1 M NaOH or KOH). The centrifuge the non-soluble material. If you are just doing a e.g. DI H2O extract you won't be getting all the humic substances out. However, if this is a good way to get the proteins out you can than acidify the extract to pH 1. The HA will precipitate and can be removed by centrifugation. You can then apply the resulkting to fulvic fraction to the DAX-8 column which will bind the FA. The material that comes through will contain lower molecular weight organic acids/phenols e.g. carboxylic acids. Some of these can be removed by applying to the XAD-4 resin. The XAD-4 resin is not used to capture HA/FA because, for the most part their molecules are too big and will not go through the resin. You will have to experiment and see what your recovery of proteins are at each stage.
google: XAD-8 XAD-4 and Leenheer to get some papers detailing these procedures. Good luck! Check out this paper: Article Quantitative evaluation of XAD-8 and XAD-4 resins used in ta...
If you just bought the resin, you have to first soak in methanol to wet it up well and remove the really fine fines. The will float on/near the surface and can be scooped off. Then rinse with DI H2O to remove the methanol. This is best done in batch mode in a large plastic/glass beaker. I use a 4L beaker. Then load into the column and continue to rinse with DI H2O until all the methanol is removed. Then you pretreat the resin with dilute HCl applied to the top of the column. Then you rinse out the acid with DI H2O applied through the bottom of the column. This procedure is detailed in my paper "A New Standardized...." free on my RG site. Remember you need to acidify the FA fraction prior to application to the resin if have not already.
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