Precepitation during dialysis - (Aug/20/2006 )
I am dialysing my protein (50 mM NaH2Po4, 300 mM NaCl, 250 mM Imidazole Ph 8.0) against
buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 Ph8.0) and changing my buffer twice one after 3-4 hrs. But there are some white precipitates and I think my protein is forming precipitate.
How to avoid these precipitate.
regards
At first glance I suppose that the precipitates were mainly Calciumphosphates which are known to be hardly soluble especially at 4°C. I suggest to start dialysis without or with much lower concentration of CaCl, and add over a long term Calciumchlorid-solution dropwise. If you sure that really protein is precipitating, it may be strongly phosphorylated and precipitates with CaCl.
i agree with kosmodrom that the precipitate is caphosphate. if you are losing protein, keep in mind that caphosphate adsorbs protein.
buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2 Ph8.0) and changing my buffer twice one after 3-4 hrs. But there are some white precipitates and I think my protein is forming precipitate.
How to avoid these precipitate.
regards
hi samita
yes, hydrated calcium phosphate is a good protein binder, and can bring down your protein.
normally, when i have to switch buffers of the sample, i first dialyze it against distilled water and then against the second buffer. i do not know if this will harm your protein, but if your protein can withstand being in distilled water for some time, it might be a good idea to do this.
many times, several proteins tend to get denatured during dialysis and are removed by centrifugation. are you sure its your protein that is going down, if not, then just centrifuge the contaminants away.
- viv
I centrifuge the precipitat and run sds gel of just supernatent and pellet. I found the band in both. So it mean its calcium phosphae and i am using just CaCl2 not CaCl2.H2O.
So next thing that i can do is i will dialyse my protein againt distelled water and let see if its stable or not.
With Buffer as well but CaC2. drop wise but it will not affect the pH.????
regards
samita
Dialysing against distilled water may become critical as you have no buffer system (distilled water does not mean necessarily that you are working with neutral pH (acidification by CO2)) or ionic strenghth which both force denaturizing; if you work with your buffer system, dropwise addition of CaCl2 up to low mM should not be critical to pH
Some proteins become less stable in the absence of salt. Dialysis against 20 mM Tris-HCl, 50 mM NaCl initially, then switch to 20 mM Tris-HCl, 50 mM NaCl plus CaCl2 as suggested by kosmodrom.