Antibody requirements

Antibody requirements

For conjugation with Proseek Probemaker, use either one single polyclonal antibody batch or two matched monoclonal antibodies. The antibody or antibodies should meet the following criteria:

•    If a polyclonal antibody is used, it must be raised against the whole native protein (not peptides) and be antigen affinity purified (not IgG fraction).
•    The antibody must have a concentration of 1 mg/ml (10 µl is needed per conjugation).
•    The antibody has to be in an amine free buffer, ideally PBS. The buffer should be carrier free but may contain up to 0.1% BSA, 5% trehalose and 0.02% sodium azide.

If you are unsure about the composition of the buffer your antibodies are stored in, Olink strongly recommends dialysis or buffer exchange prior to conjugation. Antibodies from various sources and suppliers vary in residual primary amine content often in the form of remaining glycine from the acidic elution of the antibody during the antigen-affinity purification step. Even if the supplier states PBS content, it is likely that there are residual amounts of primary amines. Antibodies from R&D Systems are suitable to conjugate directly with Proseek Probemaker and do not require dialysis.

Olink recommends the following standard procedure for buffer exchange:
Pre-equilibrate an illustra MicroSpin G-50 Columns spin column (Art no. 27-5330-01, GE Healthcare) with 1xPBS by first spinning the column at 3000 rpm for 1 min, then add 400 ul of 1xPBS and spin again for 1 min and repeat 4 times. Place the column in a new microfuge tube.
Add your antibody (25-50 ul) to the column and spin again for 1 min at 3000 rpm. The concentration of the collected antibody should be verified by OD. 1 mg/ml should have an OD 280nm of 1.4.
Concentrating low concentration antibodies prior to Proseek Probemaker conjugation is not recommended unless you have large milligram amounts since losses are very high with filter type concentrations.

Proseek probes from one Probemaker conjugation will be sufficient for approximately 100 000 reactions.