Phosphate buffers - protein and NaCl affecting pH of buffer (Apr/08/2005 )
My protein is happy in 50mM NaPO4 pH8.0 250mM NaCl. I am trying to adjust the pH in a few steps so I can do an NMR experiment on 5 pHs between 8.0 and 6.6. There is a published spectrum at pH6.6 in 10mM PO4 and 250mM NaCl. The theoretical pI of the protein is 6.8(!)
I've been told salt and protein can buffer the solution, it would be easier to change pH in a 10mM PO4 rather than 50mM PO4 solution and moving the solution to an eppendorf would allow the solution to mix more easily. Does anyone know if these things are true or the best way to adjust the pH?
I have as a guide 250ul 0.9mM protein in an NMR tube and I do not want to change the volume/concentration much
I am not sure how best to adjust the pH to minimise protein precipitation. I have tried the following:
-10M HCl causes very localised pH changes and I lose some protein.
-I lowered the phosphate to 10mM but when I added 5ul of 1M HCl the pH dropped dramatically and my protein turned almost instantly solid and I now have about 0.1mM protein :-(
(I'm wondering if it was really 1M HCl as the label said?)
-it's been suggested I add NaH2PO4 as it will be more gentle on the protein, although the amount I calculated to add seemed impractical either several mls or too small to weigh out (maybe would work for 50mM?)
-I could dialyse each time, but each NMR experiment is about 20 hours
-after staying v late tonight after my protein solidified I just tried changing buffer using a vivaspin
Maybe you can try Amicon ultrafiltration to exchange your buffer and reconcentrate your protein. I use Amicon Ultra-15 (for 15ml volumes).
I've been told salt and protein can buffer the solution, it would be easier to change pH in a 10mM PO4 rather than 50mM PO4 solution and moving the solution to an eppendorf would allow the solution to mix more easily. Does anyone know if these things are true or the best way to adjust the pH?
I have as a guide 250ul 0.9mM protein in an NMR tube and I do not want to change the volume/concentration much
I am not sure how best to adjust the pH to minimise protein precipitation. I have tried the following:
-10M HCl causes very localised pH changes and I lose some protein.
-I lowered the phosphate to 10mM but when I added 5ul of 1M HCl the pH dropped dramatically and my protein turned almost instantly solid and I now have about 0.1mM protein :-(
(I'm wondering if it was really 1M HCl as the label said?)
-it's been suggested I add NaH2PO4 as it will be more gentle on the protein, although the amount I calculated to add seemed impractical either several mls or too small to weigh out (maybe would work for 50mM?)
-I could dialyse each time, but each NMR experiment is about 20 hours
-after staying v late tonight after my protein solidified I just tried changing buffer using a vivaspin
Hi there,
The best way for the protein would most probably be to alter the pH with combination of NaH2PO4 and Na2HPO4. These buffers should be mixed together to get the right pH (no adjusting with HCl). How critical is it to know the phosphate conc.? If not important use 10mM of buffer and lower pH with 100mM NaH2PO4 (for example). Can't you make different samples for each pH? My knowledge with NMR is VERY limited so please excuse these comments if they don't make sense....
Ben
hi
i agree with Obi-Wan Kenobi. NaH2PO4 and Na2HPO4 solutions can make very good bufffers ranging from 6 to 9 (depending on amount of each basal solution used for make the mix).
So i would recommend you to do two solutions of 50mM NaH2PO4 and 50mM Na2HPO4 and mix them to obtain a solution at pH6.6. Then add NaCl powder to get 250mM NaCl and dialyse your protein against this buffer.
fred
hi
i agree with Obi-Wan Kenobi. NaH2PO4 and Na2HPO4 solutions can make very good bufffers ranging from 6 to 9 (depending on amount of each basal solution used for make the mix).
So i would recommend you to do two solutions of 10mM NaH2PO4 and 10mM Na2HPO4 and mix them to obtain a solution at pH6.6. Then add NaCl powder to get 250mM NaCl and dialyse your protein against this buffer.
fred