Protein Purification - Problem in protein purification (Jul/25/2006 )
Hi!
I am trying to purify a nitrilase protein (soluble protein). I used the cell free extract for PEI treatment (where 2X PEI=0.25% PEI, 3% NaCl, 100mM Borax, pH=7.4) and 1:1 (volume/volume) PEI was used for the lipid precipitation at this stage. Next I want to salt out my protein with ammonium sulfate. I have seen my protein band (36.2KD) in cell free extract in SDS gel but surprisingly did not find any band for my protein after PEI treatment. Even I have tried 1X PEI treatment but it didn't help. I have used similar procedure to purify other Nitrlase very similar to it and our lab use almost the same procedure for all other protein purification like ketoreductase, aminoacid dehydrogenase etc. I had no problem with this before for all of my other protein , mostly nitrilase.
If anybody could help me with this problem i will be much thankful.
cmsk
It may form a complex with PEI (a basic polymer) through charge-charge interaction. Is it anionic charged at the pH that you are working with? PEI treatment can be tricky at times.
How can i avoid this complex formation? I do not know if it is anoinic or not at this pH. Basically I am a Organic chemistry student and trying to make some Nitrilase enzyme for synthetic pursose.
Tahnk you
It may form a complex with PEI (a basic polymer) through charge-charge interaction. Is it anionic charged at the pH that you are working with? PEI treatment can be tricky at times.
How can i avoid this complex formation? I do not know if it is anoinic or not at this pH. Basically I am a Organic chemistry student and trying to make some Nitrilase enzyme for synthetic pursose.
Tahnk you
Why are you using PEI in the first place? Can you not simply ammonium sulphate precipitate?
It may form a complex with PEI (a basic polymer) through charge-charge interaction. Is it anionic charged at the pH that you are working with? PEI treatment can be tricky at times.
How can i avoid this complex formation? I do not know if it is anoinic or not at this pH. Basically I am a Organic chemistry student and trying to make some Nitrilase enzyme for synthetic pursose.
Tahnk you
Why are you using PEI in the first place? Can you not simply ammonium sulphate precipitate?
It is necessary to remove the neuclic acids and lipids part from my lysate otherwise it would stuck in the diafiltration membrane. I have tried this before and ruined two of those membrane so I am afraid to do so now.
Can you use ultracentirfuge to remove these ? What organism are you working with?
Have you tried adding some DNase I to the lysis buffer? That will get rid of the nucleic acids.
What exactly is your purification strategy?