i want to ask about suitable purification protocol for antibody from sodium azid or proclin 300 to use antibody for conjugation with magnetic nanoparticles and gold nanoparticles.
Should i do purification before dividing the antibody into aliquots for storage or i can store it at -20 then apply purification prior to use.
What about using NAP 5 column for antibody purification?
Yes, you can use NAP-5 columns. While they seem to be promoted for DNA work, they contain Sephadex G25M, which is a standard desalting medium for cleaning up small volumes of antibody. I've used hundreds of them for this purpose.
Whether you desalt first or second depends on the size of aliquots that you plan to store, because you will need at least 100ul, preferably at least 200ul, to load onto the NAP-5 column. Personally, I do 200ul though the manufacturer claims you can do more than this and still get good separation.
An advantage of desalting all your antibody first is that all stored aliquots will be identical.
If you have too much antibody to load onto a NAP-5, you could use the larger PD10 column (I load 1-2ml on these columns).
However, if your volume of antibody is greater than 10ml it may be more efficient to do dialysis than desalting on multiple columns.
I'd desalt the antibodies in large batches (at least 1mg, better more). Otherwise, you'll lose substantial amounts which bind to the column and by peak widening. If you only have small amounts, use the smallest column size that fits your volume. If the antibodies are dilute (below 1mg/ml), consider concentrating them.
Re-using the columns is fine, at least for the same antibody. Just equilibrate them with at least 5 column volumes to wash out all the salts. For storage then use PBS with a little sodium azide to prevent microbial growth.
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