What dialysis buffer should I use for the protein subsequently to be used for ki - What decides the composition of the dialysis buffer? (Jul/31/2007 )
Hi folks,
I plan to purify protein (GST-fusion kinase) and subsequently to do in vitro kinase assay.
After purification step, what dialysis buffer do I have to use? For the in vitro kinase assay, I need to use HEPES. Do you think it is better use HEPES as dialysis buffer? or what about PBS? May phosphate groups in the PBS interfere with kinase reaction somehow? Has anyone ever used PBS for dialysis and done kinase assay subsequently? Please share your information. Thank you!
-cancerous-
QUOTE (cancerous @ Aug 1 2007, 12:37 AM)
Hi folks,
I plan to purify protein (GST-fusion kinase) and subsequently to do in vitro kinase assay.
After purification step, what dialysis buffer do I have to use? For the in vitro kinase assay, I need to use HEPES. Do you think it is better use HEPES as dialysis buffer? or what about PBS? May phosphate groups in the PBS interfere with kinase reaction somehow? Has anyone ever used PBS for dialysis and done kinase assay subsequently? Please share your information. Thank you!
I plan to purify protein (GST-fusion kinase) and subsequently to do in vitro kinase assay.
After purification step, what dialysis buffer do I have to use? For the in vitro kinase assay, I need to use HEPES. Do you think it is better use HEPES as dialysis buffer? or what about PBS? May phosphate groups in the PBS interfere with kinase reaction somehow? Has anyone ever used PBS for dialysis and done kinase assay subsequently? Please share your information. Thank you!
PBS is good to save existing phosphorylations but, to my knowledge, does not interfere with kinase action;
HEPES is better but more expensive
-The Bearer-