Hmmm ... I think binding of the protein should be the same as long as both buffers have the same pH, which requires the concentrations of both buffers to be sufficiently high to properly maintain the pH (I generally use 0.1 M).

You also mention the salts in the two buffers are different. Do you mean both buffers have different concentrations of the same salt or different salts but with the same final concentration? Different salts behave differently and since elution of the protein from an ion exchange column depends on the salt, both of these scenarios could result in different elution profiles.

What temperature did you run the columns at? The pH of TRIS changes with temperature. This means that if you prepare the buffers at room temperature they will both have pH 8.2, but if you then run the column in a cold room (to protect the protein from degrading) the pH of the TRIS buffer will be different from that of the HEPES buffer.

Anyway, if your purification works fine in TRIS why change it to HEPES? If for some reason you do need the protein to be in HEPES for your assays, I would first purify it in TRIS and then dialyse the purified protein into HEPES afterwards.

Hmmm ... I think binding of the protein should be the same as long as both buffers have the same pH, which requires the concentrations of both buffers to be sufficiently high to properly maintain the pH (I generally use 0.1 M).

You also mention the salts in the two buffers are different. Do you mean both buffers have different concentrations of the same salt or different salts but with the same final concentration? Different salts behave differently and since elution of the protein from an ion exchange column depends on the salt, both of these scenarios could result in different elution profiles.

What temperature did you run the columns at? The pH of TRIS changes with temperature. This means that if you prepare the buffers at room temperature they will both have pH 8.2, but if you then run the column in a cold room (to protect the protein from degrading) the pH of the TRIS buffer will be different from that of the HEPES buffer.

Anyway, if your purification works fine in TRIS why change it to HEPES? If for some reason you do need the protein to be in HEPES for your assays, I would first purify it in TRIS and then dialyse the purified protein into HEPES afterwards.

If the pI of the protein is 6.5 and the pH is 8.2, the protein is predominately negatively charged and you will probably get better purification with an anion exchange column (support is negatively charged) rather a cation exchange (support is negatively charged).

Tris is near neutral at pH 8,2 (pKa 8.1). Hepes is negatively charged (pKa 7.55). Hepes buffer has a greater number of cations (since there must be an equal amount of Na+ to balance Hepes-). If the buffer concentration  is less than or nearly equal to the salt concenration, the higher ionic strength of the Hepes buffer is probably displacing your protein from the column.

If the salt concentration>> than the buffer concentration, then the ionic strength of the buffer is probably not affecting the buffer much. In that case you probably have a specific interaction of Hepes with the protein which cannot be displaced by the column media.

The solution in either case is the same. Either use an anion exchange column (a different set of residues is involved so another set of  specific interactions is unlikely) or purify it in Tris then dialyze it out as suggested.

Obviously the buffer you use has an impact on the behavior of your protein. If you provide precise buffer composition, it should be easier to discuss and to find what the problem is...

The final pH of TRIS buffers are very temperature sensitive.  Thus, if you measured pH 8.2 at 25 deg C (room temperature), but performed chromatography at 4 deg C, the actual pH would be approximately 8.8!  HEPES is much less temperature sensitive.

See https://www.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer.

Hello Manjula,

A few thoughts differing from the other answers.

1) Did you prepare theTris-HCl and HEPES buffers used in yoru chromatography from mother solutions ? Upon dilution, Tris-HCl buffer change its pH quite impressively sometimes. So, if the pH82 was measured on the mother buffer, the diluted one has a different pH !

2) Tris is a base with a primary amine, HEPES is an acid molecule with a piperazine skeleton and a sulfonic acid group ... these molecuels are NOT necessarily "neutral" (if I may say so) in respect with their binding to the enzymes you wish to purify, they can be ligand of your enzymes yielding to modification of the binding property of your protein to the column ...

there are other possibilities ...

Bets reagrds and very good luck !

Philippe

Hi Manjula, just to add to Jeffrey's answer: HEPES has a buffer range of 6.8 - 8.2. Tris has a buffer range of 7.5 - 9.

HEPES is not a good buffer at the pH that you are using. It is at its limits at pH 8.2.

Effectively, HEPES at  pH 8.2 is probably acting more like a salt than a buffer and is preventing binding to the Q column.

Hi, 

Although it would be late, but maybe helpful for other people looking for answers.

HEPES is NOT suitable for anion exchange chromatography (but suitable for Cation Exchange instead), while Tris is.

Please read this manual for confirmation:https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1454593346687/litdoc11000421_20160204144201.pdf

Thank you.

HEPES  Stable pH vs. temperature, no primary amine groups, no metal chelation, near physiologic pH range. HEPES is often used to maintain protein solubility in biochemical experiments.The activity increased in the following order: citrate < Tris = potassium phosphate< HEPES. presence of more than 10 mM HEPES at pH 8.0 was attributed to the surfactant activity of this buffer,, HEPES and Tris, being positively charged at pH 7.0, were not bound as readily as phosphate. oligonucleotide initiator decreased in the following order: cacodylate> MES >HEPES> TRIS >phosphat.The presence of as much as 100 mM NaCl increased the stability and reversibility of unfolding transitions in Hepes buffer but not in phosphate buffer at pH 7.4

HEPES interferes with the Lowry protein assay (not the Bradford assay). It can form radicals under various conditions and should not be used in systems where radicals are being studied.

Tris possesses a potentially reactive amine and participates in various enzymatic reactions. The pH of a Tris buffer is affected by the temperature (see above) and the concentration. The pH decreases 0.1 unit upon a tenfold dilution.

https://www.researchgate.net/publication/309041540_Important_Factors_Influencing_Protein_Crystallization

Article Important Factors Influencing Protein Crystallization