I performed purification on FPLC hydroxyapatite column (CHT5-I from Bio-Rad) last week. I attached the chromatogram. Running conditions were as follows:
buffer A: 5 mM KH2PO4; pH 8.0; 0.15 M NaCl; 5 mM mercaptoethanol; 0.5 mM CaCl2
buffer B: 0.75 M KH2PO4; pH 8.0; 5 mM mercaptoethanol
the sample was in 50 mM Tris/HCl; pH 8.0; 0.15 M NaCl; 5 mM mercaptoethanol.
I managed to collect separately the very first peak, than the three smaller peaks together and the main peak at the beginning of the gradient. The activity was both in the three peaks and in the peak at the start of gradient. I'm looking for any suggestion, how to improve the binding, because it obviously eluted everything very early.
My ideas:
1) Use lower pH of the buffers, but I'm a little scared of that since my protein likes much more higher pH (9-10). And I'm not sure, if it would really help, but it seems that everybody is using pH between 6.5-7.5
2) exchange the buffer of the sample. However, I tried to keep the Tris buffer with other enzyme once and there was no big difference.
The fact you are getting some protein in what looks like the flow through suggests you may have overloaded the column. Do you get the same profile if you load say half as much? Alternatively, if it is the wash, it might be due to the phosphate. You could use an alternate buffer as buffer A (with 1 mM phosphate). Maybe try omitting the NaCl too? Salt can be used to elute proteins from HA. Why is the CaCl2 there in buffer A - if it isn't required, drop it out as well. Calcium does change the binding properties of HA resins.
If that doesn't work, you could try a different HA resin, or play with the pH. I've generally used a slightly acidic buffer. You can always collect into tubes with a concentrated high pH buffer to re-set the pH once the fraction is collected.
Or maybe you just have different forms of the protein, and they bind differently?
I am afraid to point this out, your buffer A has only KH2PO4 and missing (its conjugate base) di-potassium/sodium phosphate(may be you forgot to add here?). Please have a look at the phosphate buffer preparation. You know, phosphate buffer should not be prepared like HEPES/Tris or any other buffer.
Capacity of the column is 50 mg of proteins. I loaded only about 1 mg or so, so it was not overloaded for sure.
Yeah, I've seen it somewhere recently, that proteins can be eluted from HA by salt. Didn't know about that before. However, I'm little scared to get rid of it because of my protein. But I'm using no-salt-buffer for anex chromatography anyway.
I was told, that addition of CaCl2 improves binding in combination with low phosphate concentration.
Sure they could be different forms, but why the bound proteins elute only as single peak?
The buffer was titrated with KOH to desired pH.
Don't be afraid to cut out the NaCl in the chromatography buffers. A KPO4 buffer should provide all the ionic strength you need. Also, try to decrease the pH and worry about the protein quality if you actually do have problems with it after the purification. Protein sticking to the matrix can be fine at a pH the protein does not like in solution.
I never added CaCl2 in HA chromatography buffers, but I would recommend for the next trial:
Buffer A) 100mM KPO4 pH 7.0, 5mM BME
Buffer B) 700mM KPO4 pH 7.0, 5mM BME
Don't titrate KH2PO4 with KOH unless you want to buffer around pKa3 (~12).
Hi Tomas--I'm one of the technical experts on CHT at Bio-Rad. E-mail me if you're still having problems and I can help.
Mark A. Snyder
OK, I omitted the CaCl2, prolonged the gradient and decreased pH of the buffer to 7.0 (and added glycerol since it's crucial for the protein stability). There was some improvement visible, but the bound proteins still eluted basically in one peak.
If I'l ever get back to the project (which is unfortunately not sure now), I'll try to decrease the pH some more and prolong the gradient even more, but I'd want the proteins to bind stronger because under my conditions they elute literally in the second the gradient starts.
Can somebody explain to me, why I cannot titrate the phosphate to desired pH? There is the equilibrium of particular ions and in my way the molarity is exact.
Proteins have multiple different interactions with hydroxyapatite including cation exchange, metal chelation, anion exchange and hydrogen bonding. This was systematically examined with a library of proteins. The results suggested synergistic binding with both metal chelation and cation exchange interactions play a major role for protein binding. So I would take a look at Anal Chem. 2011 May 15;83(10):3709-16. doi: 10.1021/ac103336h http://pubs.acs.org/doi/pdf/10.1021/ac103336h. For phosphate buffer titration - because phosphoric acid has multiple dissociations you might try a phosphate Buffer Calculation (Javascript) - Fuse Home Pages
home.fuse.net/clymer/buffers/phos2.html - A Javascript that calculates the amount of monosodium phosphate and disodium phosphate necessary to achieve a buffer at a given pH and buffer strength.
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