Quasi26 at concentric.net wrote:
>> I am a student at WPI in Mass. and was wondering if you could help me
> purify this protein.
>> The protein has a MW of 6,000, pI @ 4.9 and is intracellular
> The contaminants have MW of 113,000; 13,000; and 39,000
> Acetate is an inhibitor and the protein is being used for the food
> industry
> It is good at temp. from 22 - 62 C, with the highest activity between
> 40-50 C
> It is good at ph from 4 - 10, with high activity between 6 and 9
>> I am just looking for a simple scheme, using tangental flow, ion
> exchanges, HIC, Gel filtration, ultra, PPT etc...
>> This is a scheme that I have chosen but I was wondering if you could
> help me get more in depth with it or show something that would work
> better. I use simple flow sheets, but I will write this out because I
> can not think of a way to make a flow sheet on this.
>> From the cells, I choose to centrifuge and then break them (French
> Pressure cell), Succinate buffer (pKa of 5.6). From the homogenate, I
> choose ultrafiltration, using 100K at cutoff (getting rid of 113,000).
> then discard pellet and take your low level supernatant and use cation
> exchanger with succinate again. pool the active fractions and maybe
> use Sephadex G 25 or HIC.
>> This is just a simple scheme, but I was hoping that you could critique
> it and help me on it. I am taking a purification lab next term, and
> this was given to us to show us what we will be seeing in the future.
> I also have another question, What ph range should I operate from and
> how do you determine that?
>> Hope to hear from you soon. My email is davido at wpi.edu Thank
> you.
I would say, try it, looks good. I don't know how stable your protein
is, but if it is not easily deactivated by the proteases in your
homogenate, try using a lower cut-off ultrafiltration, e.g., 10000 or
20000.
Another possiblility: skip the ultrafiltration step and go directly to
the cation exchanger. If the homogenate is too turbid because of
colloidal particles, use a 'Big Beads-material' with a larger pore-size;
we do this routinely, I am not quite sure, but I think we buy the gel
from Pharmacia.
Achim
