Me and my partner are doing project involving the purification of equine IgG using both anion and cation exchange chromatography. The main purpose of our project is to separate IgG from other protein plasma. The matrix that we use are weak anion exchanger Fractogel® DEAE (pre-packed column), and weak cation exchanger Fractogel® EMD COO-. In the condition of pH 6, the purification of IgG showed much better separation using anion exchanger where IgG (pI = above 6) is eluted in the flowthrough fractions, whereas the contaminants (mostly albumin with pI around 5) are bound to the matrix and eluted using salt gradient concentration method. SDS-PAGE also confirmed that the flowthrough fractions contain only single band IgG with MW around 150 kDa.
However, during the experiment with cation exchanger in the same pH condition, contaminant albumin which is supposed to elute in the flowthrough fractions is also bound to the matrix and eluted together with the target protein IgG. What caused the albumin to elute with IgG, where in theory, albumin should have negative charge and should’ve not bonded to the matrix?
Equilibration buffer (Buffer A): 20 mM phosphate buffer pH 6
Elution buffer (Buffer B): Buffer A + 1 M NaCl pH 6
There are several reasons, including checking the pH of the buffer or The matrix in the used column Or a change occurred in the nature of the protein during the purification process
Dear Shanaz
the difference in terms of pI from your igG and the contaminants (5 vs 6) is not so high and tuning of cationic and anionic exchange is not so fine, also because the pI is predicted on the basis of linear aminoacidic sequence but is it possible that in reality the folded protein and antibody expose in the surface different charged and hydrophobic residues and real pI is different from the theoretical.
I suggest to you to use proteinA and proteinG resins that show higher and specific affinity for igG.
Those resins are expensive, but it have high binding capacity (5-10mg/ml) therefore you can use very small volumes manually packed in gravity flow columns and you can re-use it several times.
https://www.sciencedirect.com/science/article/abs/pii/S0165242796056188
good luck
ciao
Manuele
Hi there,
Albumin is a naturally sticky protein which means its behavior towards chromatography matrix doesn't always obey to the theory.
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