Elute fractions that have target protein in FPLC is unstable - (Dec/23/2014 )
Hi everyone,
I'm purifying recombinant growth factor. It's extracellular protein. The isoelectric point of protein is 9.8, so I use elute buffer at pH 7.5.
I have done many times, 15 approximately. But, I don't understand what happen. The result isn't stable. Protein is eluted at 60% B buffer. Another times, It start at 70%... And the cleanest fraction is unstable, too. I see it cleanest at 70, 75, sometimes at 80, 85.
I'm confused with my experiment. If you know anythings, please help me.
Thank you so much.
using which matrix are you attempting the purification?
do you clean the matrix after each run?
do you regenerate?
mdfenko on Tue Dec 23 22:40:48 2014 said:
using which matrix are you attempting the purification?
do you clean the matrix after each run?
do you regenerate?
I use Hitrap SP FF column 5ml. After each run, I use NaCl 2M, acetate buffer 20mM pH4.0 to clean the column, then storage in NaOAc 0.2M, EtOH 20%.
My column is reused many times, upper 20 times.
do you prepare the buffers fresh each time?
what is the composition of buffer b?
how do you determine that your column is properly equilibrated prior to loading the sample?
if the pH of your buffer drifts up then the protein should elute earlier in the gradient.
mdfenko on Wed Dec 24 21:29:55 2014 said:
do you prepare the buffers fresh each time?
what is the composition of buffer b?
how do you determine that your column is properly equilibrated prior to loading the sample?
if the pH of your buffer drifts up then the protein should elute earlier in the gradient.
I make a large flask buffer, so I use for many times. B buffer has NaCl 1M, Tris-HCl 20mM pH 7.5. Before I loaded the sample, I washed the column by dH2O until conductivity reach 0% and stable. Then I equilibrated by 15ml NaOAc 20mM pH 4.0.
it may be that your pH isn't stable during the elution.
you are running a pH gradient as well as a salt gradient during elution. the pK's of acetate and tris are far enough apart that you will not have sufficient buffering capacity to stabilize the pH during the run.
as the buffer ages, more and more co2 will dissolve in the solutions, thus altering the elution profile.
you may want to try preparing fresh buffers when yo run or use another buffering salt (either replace or supplement).
mdfenko on Sat Dec 27 00:49:36 2014 said:
it may be that your pH isn't stable during the elution.
you are running a pH gradient as well as a salt gradient during elution. the pK's of acetate and tris are far enough apart that you will not have sufficient buffering capacity to stabilize the pH during the run.
as the buffer ages, more and more co2 will dissolve in the solutions, thus altering the elution profile.
you may want to try preparing fresh buffers when yo run or use another buffering salt (either replace or supplement).
Thank you, I'll try.
trungnghiatn on Thu Dec 25 03:02:46 2014 said:
I make a large flask buffer, so I use for many times. B buffer has NaCl 1M, Tris-HCl 20mM pH 7.5. Before I loaded the sample, I washed the column by dH2O until conductivity reach 0% and stable. Then I equilibrated by 15ml NaOAc 20mM pH 4.0.
I would also look to replace the Tris with HEPES when using a cation exchange resin. Tris ions will bind to the SP group altering the buffer capacity.
as the buffer ages, more and more co2 will dissolve in the solutions, thus altering the elution profile.
Agree. Moreover, if your system does not have a degasser you should not used aged buffers or at least degas them before every use.
You should also pay attention to conductance, not buffer concentration at which your peak appears. The buffer concentration informs you about the "input" - how pumps work - while conductance informs you about the output and better pictures what really happens during the run.