Anti-PTM antibodies can not be purified using a classic immunopurification procedure, for control peptide-specific antibodies have an a non-negligible affinity for the modified peptide. Indeed, immobilising the modified peptide onto a column and loading the antiserum directly would lead to the binding of a large quantity of antibodies directed against the unmodified peptide, leading to a dramatically reduced specificity for the expected post-translational modification.
Our 3-step purification procedure allows us to specifically separate the antibodies which are directed against the modified peptide from those which are directed against the control one.
The immune serum is first loaded onto a column in which the control peptide (yellow) has been previously immobilised on the agarose beads. Therefore only the antibodies which are specific for the control peptide are retained while all other molecules including antibodies of interest are discarded in the flow-through.
The flow-through is subsequently loaded onto another column in which the modified peptide (green) has been previously immobilised so that the antibodies of interest are this time retained within the column while other unwanted molecules are discarded in the flow-through.
Highly pure modified peptide-specific antibodies are finally eluted from the column.
In order to control that the final solution of antibodies are indeed specific for the modified peptide and not for the control one, ELISA are performed against both control and modified peptides.
A complete data report which summarises the conditions of purification and showing the ELISA results are provided with the antibodies. Antibodies are provided in a Tris-glycine buffer (glycine 1 M, Tris 0.1 M, pH 7.8) containing 0.02% sodium azide. If you do not wish azide to be added, please inform us while placing your order.
Figure: anti-PTM antibodies purification procedure