I have problems to purify an IgG1 Mab. How I can do it? The concentration in protein A pH6 is very low
If you are purifying mouse IgG1, they bind to Protein A at pH 8 but start dissociating at pH below 7. Other mouse subclasses or rabbit IgG bind more stably to Protein A. So for purification of mouse IgG1 be sure the pH of binding buffer is 8 or higher; for elution you may use a mildly acidic buffer, pH 5 or so. Alternatively you can think of other resins, as Viktor suggests, or Protein G.
You can try MEP-HyperCel resin, it is high capacity sorbent with very mild conditions for elution. You can load your sample in any buffer with pH 6.0 or higher even at very low Ab concentration.
http://www.pall.com/main/biopharmaceuticals/product.page?id=35677
If you are purifying mouse IgG1, they bind to Protein A at pH 8 but start dissociating at pH below 7. Other mouse subclasses or rabbit IgG bind more stably to Protein A. So for purification of mouse IgG1 be sure the pH of binding buffer is 8 or higher; for elution you may use a mildly acidic buffer, pH 5 or so. Alternatively you can think of other resins, as Viktor suggests, or Protein G.
Protein A column could be used for purification. I agree with Marco.
We use 1M Glycin/NaOH + 0,15 M NaCl pH 8,6 for loading and washing of the column. Elution with citric acid buffer try several 0,1 M Citric acid pH 6, 5, 4 or 3. Start with the highest one and monitor UV. ProSep ultra plus columns with protein A have the advantage of not being sensitive to running dry.
If it is valuable antibody for you, it is simple to use Amersham Hi-Trap PA column, depend on the concentration of the antibody you can use 1ml or 5 ml fast flow. it is very simple to use syringes, peristaltic pump, or automatic machine for loading the sups or sera and eluting the IgG according to the manufacturer procedure. Still if you do not want to use the column, have a look on the manual.
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