I am working on a project where I have to purify some proteins that are tagged with a 6x His tag. Since my proteins were produced by bacteria, I purchased the HOOKTM 6X His Protein Purification (Bacteria): Nickel Resin kit.
The proteins that I am purifying are proteins that crosslink each other to form a coagulant and require calcium to be activated. Unfortunately, I found out that the His Elution Buffer that comes with the kit reacts with calcium to form a precipitant. The His elution buffer is made of the following ingredients: 250mM Imidazole, 300 mM NaCl, and 50 mM Sodium phosphate (pH 8.0).
I don't know if that is supposed to happen or not, but that means that I can't see if my proteins are actually getting activated by the calcium and crosslinking.
Does anyone know why this might be happening?
Am I allowed to use a different buffer to elute my His tag purified proteins?
The problem is that Calcium phosphate is insoluble. Calcium in your protein is exchanging with the sodium phosphate in your elution buffer to form calcium phosphate. Pretty much all phosphate salts are insoluble at any meaningful concentration. Don't use phosphate in your buffer.
Assuming you have Tris powder, Sodium Chloride, Imidizole flakes, and concentrated HCl, and a pH meter on your shelf on your shelf you can make your own elution buffer:
250 mM Imidizole, 300 mM NaCl, 50 mM Tris-HCl (pH = 8)
Say you want to make 1 L of solution:
grams of Tris = (0.050 moles Tris / L) * (121. 14 g Tris / 1 mole Tris) * 1 L = 6.057 grams of Tris
grams of Imidizole = (0.250 moles Imidizole / L) * (Formula weight Imidizole) * 1 L
grams of NaCl = (0.300 moles NaCl / L) * (Formula weight NaCl) * 1 L
Add the required amounts to a 1 L beaker with stirring with a magnetic stir bar. Add 800 mL of deionized water.
Calibrate your pH meter before measurement. Adjust pH by adding HCl dropwise to form Tris-HCl and lower the pH to 8. If lower the pH too much add concentrated NaOH to bring the pH back up to 8. Then add more water to fill your beaker up to the 1 L mark.
Then maybe you want to filter your buffer. Use this now.
Three possible solutions:
1) Dialyze your protein against another buffer (Tris, Hepes etc. ) after elution. This is probably a good idea anyway to get rid of imidazole.
2) Elute your protein with a Tris buffered solution (e.g. Tris-HCl 50 mM pH 8) containing NaCl and imidazole
3) Decrease the amount of Ca (add it from a dilute solution ≤ 100 mM) so that you don't overshoot the solubility product of calcium phosphate. I don' know the Km of your protein for Ca2+, but 10exp-4 M Ca2+ probably won't precipitate and may still be sufficient to activate you protein
The problem is that Calcium phosphate is insoluble. Calcium in your protein is exchanging with the sodium phosphate in your elution buffer to form calcium phosphate. Pretty much all phosphate salts are insoluble at any meaningful concentration. Don't use phosphate in your buffer.
Assuming you have Tris powder, Sodium Chloride, Imidizole flakes, and concentrated HCl, and a pH meter on your shelf on your shelf you can make your own elution buffer:
250 mM Imidizole, 300 mM NaCl, 50 mM Tris-HCl (pH = 8)
Say you want to make 1 L of solution:
grams of Tris = (0.050 moles Tris / L) * (121. 14 g Tris / 1 mole Tris) * 1 L = 6.057 grams of Tris
grams of Imidizole = (0.250 moles Imidizole / L) * (Formula weight Imidizole) * 1 L
grams of NaCl = (0.300 moles NaCl / L) * (Formula weight NaCl) * 1 L
Add the required amounts to a 1 L beaker with stirring with a magnetic stir bar. Add 800 mL of deionized water.
Calibrate your pH meter before measurement. Adjust pH by adding HCl dropwise to form Tris-HCl and lower the pH to 8. If lower the pH too much add concentrated NaOH to bring the pH back up to 8. Then add more water to fill your beaker up to the 1 L mark.
Then maybe you want to filter your buffer. Use this now.
Dear Caleb
as the other suggest, the calcium and phospate are not compatible.
Is it better if you are using for IMAC buffers that contain 20mM Tris as buffer agent instead phosphate.
Generally i'm using
tris 20mM, Nacl 300mM, imidazole 10mM pH=8 as binding buffer
tris 20mM, Nacl 300mM, imidazole 300mM pH=8 as elution buffer
and buffers with intermediate imidazole concentration (eg 20-40-60mM) as washing buffer.
OF course in case you have already eluted your protein in phospate buffer, you can perform a buffer exchange by dialisys of desalting.
If you use dialisys, take in consideration that with dialysis to completelly remove the phospate and do not observe precipitation when you adding calcium is it possible that more than one step are required.
good luck
Manuele
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