Secretory universal influenza antibody CR9114 were successfully expressed by adenovirus vector ,then we used protein A G beads purify CR9114.But the molecular weight of the purified antibody under non-reduction condition seems more like a monomer containing only one heavy chain and one light chain ,which were much lower than that of the dimer.
(The molecular weight of the purified antibody were correct under reductive conditions). What's going on? Is the disulfide bond broken?
How to use protein A G beads to purify antibody?
Hello
You can try using the protocol given in the link below:
http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/antibody-purification.html
Hope this is helpful.
Regards.
The link that Malcolm Nobre provided is quite useful for Protein A/G purification.
As to the monomer issue, certain antibodies are quite easily converted from H2L2 to HL by reducing agents, so I would check the components of the medium you are isolating the antibody from to look for things like 2-mercaptoethanol, dithioerythritol/dithiothreitol or even cysteine. It is not uncommon to see HL in hybridoma supernatant, but it is not typically the predominant species present.
In other antibodies, the light chains are disulfide linked to each other, and the H2L2 form readily dissociated into H2 and L2. I don't know of any that are (HL)2 rather than H2L2, but I guess it could happen.
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