Hi Meg,
If the bigger peak is appearing when you wash with 1M NaOH, i think your
protein is precipitated on the the column. If you have your protein
relatively pure after the precedent step, maybe you can try different
buffer. You can probably find a better buffer than this one. If your
protein is in good condition before the column, maybe its a problem with
the column type. Maybe you can try an other SP. If your protein have
Cys, maybe you can try to add som DTT or 2-mercaptoethanol or something
like this.
In my opinion the best start is to detemined the better pH and after try
some other stuff like reducing agent, light detergent... (triton X-100,
Glycerol...).
Nicolas
Le 04/01/11 10:34, megha goyal a écrit :
Dear All,
We used SP sepharose high performance as second stage Ion exchange
chromatography for polishing the product. We did get pure product but
yield obtained was mere 25%. Our protein has a pI of 5.5 - 6.0 and we
had used 25 mM Na Acetate buffer pH 4.5 for loading and same buffer
with 1M NaCl for elution, 25 C.V. linear gradient. Can you suggest
some changes that i can incorporate to increase the yield i.e
additives to be added or some change in pH etc. I tried elution with
arginine HCL as elution buffer as was recommended in one paper, but
the yield obtained was even less.
On washing with 2M NaCl ther is not much peak appearing but on washing
with 1M NaOH substantial peak appears.
Kindly help me through this.
meg
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