I am trying to purify IgM from precipitate obtained from serum after dialysis against 10mM sodium acetate buffer pH 5.6. Will a 70 ml Bed volumn column will do the necessary separation to remove small MW proteins from this solublized precipitate.
Sharad,
As phrased, your question does not include sufficient information for an accurate answer. You are missing a few pieces of critical information, which are inter-related:
- How much serum you used to isolate IgM?
- What is the volume (and concentration) of dissolved IgM
- No one can guess what is your criteria of "necessary separation"
Without the above information, giving a column size means nothing.
Generally speaking, if your dissolved IgM volume is
Dear Sharad,
It is an important question, and its exact and correct answers depends on IgM concentration (mg/column?), and I can consider a bed height 10 cm and i.d 3 cm (for bed volume 70ml), but it is better give me the bed height, i.d and IgM amount estimation, for assessment adequacy of 70ml or not.
best
mehdi
I am dialyzing 50 ml of serum to get the Euglobin fraction (contains IgM and other 2-3 proteins MW
Dear Sharad, thank you for the information, I think your loading volume (1ml) is ok, I think the protein concentration is high, the best purity in your work will obtain from 5mg protein but there is no problem for 10mg. I suggest you elute with 5 column volumes of elution buffer.
best
mehdi.
what will happen to the salts will they elute with the IgM or elute separately.
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