Ion exchange Chromatography - Cation exchanger and TRIS Buffer

[Verbatim_25]

Hi People,

 

I want to purify my peptide from other byproducts. I chose Ion exchange because my peptide (Fluorophore-KARK(modficiation)SAGA) its positively charged (Lysine and Arginine). I guess that the isoelectric point is around 11 and therefore cation exchanger is more suitable than anion exchanger. The thing is that my peptide is dissolved in Tris Buffer pH 8.2 and I would like to use it also as elution buffer (Tris buffer with NaCl). Nevertheless I read that for Cation exchanger is high recommended to use Phosphate buffer and Tris is more suitable for Anion exchange. My question is Why and if the use of Tris buffer represent any problem in the cation exchanger chromatography?

 

Thanks,

[CharonY]

The binding on cation resins is usually dependent on the amino group. Check out how Tris looks like and I think you should figure it out.

[Verbatim_25]

Oh yeah. Tris when it's protonated in the amino group could compete for the anionic sites...I didn't get it before...

 

By the way,my peptide is solved in Tris Buffer and I want to start the ion exchange separation with sodium phosphate buffer. The question is, how could I remove TRIS or how can I change the buffer? do u know any simple technique to do it?

 

Thanks,

 

 

The binding on cation resins is usually dependent on the amino group. Check out how Tris looks like and I think you should figure it out.

[CharonY]

There are several ways. One could dialyse, for instance, but I prefer to use a MWCO filter and just wash it. Depends a bit on the properties of the protein, how much you can afford to lose etc.

[Verbatim_25]

Dialyse is one option, but it's time-consuming and I would prefer to use other faster techniques. MWCO filters is not really well-suited in my case since I am trying to purify one peptide that has 2000 Da as a molecular mass. It would also pass through the filter.

 

I was thinking in Desalting Columns/buffer exchange which are faster than the previous ones.

 

There are several ways. One could dialyse, for instance, but I prefer to use a MWCO filter and just wash it. Depends a bit on the properties of the protein, how much you can afford to lose etc.

[CharonY]

Most commercially columns labelled as desalting columns are actually MWCO filters. There are also MWCOs of lower than 2 kDa around. The C18 columns (which you probably are thinking of) are usually sold as spin or SPE columns (or you could use ZipTips if you only need low sample volumes). Since it appears you are doing a preparative separation you may think of a method that would already get rid of byproducts that you do not need and use this step as a prefractionation, already.

[PKLambooy]

I know this is years old but bicine is a very good buffer for use in alkaline CEX; does not interact with resin.  I published a paper on this.

https://www.sciencedirect.com/science/article/abs/pii/S1570023214006849