Alison Leakey <leakeya at citec.qld.gov.au> ecrivait (wrote):
> I was hoping for some advice to solve a problem I am having. I am
> currently trying to estimate the size of a protein with biological
> activity using size fractination. Following extraction (activity intact)
> I separate proteins using a Sephacryl S-100 column using a phosphate
> buffer with NaCl 0.15M (pH 7.0) at 4 degrees. The dilute fractions are
> subsequently concentrated using Biomax 5K centrifugal membranes.
> Activity is then measured. Unfortunately, activity is reduced by at
> least 400X by the fractionation and/or concentration. There are visable
> bands on the UV monitor readout and in silver-stained gels but no
> activity. Any advice?
> Are there any obvious things I am missing or suggestions to improve
> stability ?
I think your protein is adsorbed on the plastic (or glass) wall of
your vials.
When you purify your activity, you dilute it out from the
contaminating (and stabilizing) proteins and it sticks...
Possible solutions :
. some (...) BSA (Bovine Serum Albumin) in your recovering vials (i.e.
0.1% final concentration) or directly in your buffer (!!!) but your
protein won't be purer...
. some FCS (foetal calf (or bovine) serum) if BSA is not enough ;
. some methylcellulose in your buffer (can't remember the
concentration) but if you are interested I can find it back.
Obvious Interest : no added contaminating proteins.
I used these 3 strategies to caracterize and purify IL-2 in the early
80's. I finally ended up with reverse phase HPLC (water-acetonitrile
gradient with 0.1% TFA) after a initial phase of purification (on
silicic acid...). IL-2 activity is stable in acetonitrile.
What's the approximate MW and pI of your protein ?
Maybe you should start with a purifying method which concentrate (or
doesn't dilute too much) your sample :
. Concentrating on a membrane with a MW cutoff.
. Precipitating with ammonium sulfate
. Ion exchange eluting with a gradient
etc.
Good luck
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