In the instruction manual for the PhycoLink™ Conjugation Kits, you suggest altering the reagent proportions for my specific antibody. What happens when I reduce the amount of antibody in relation to APC?
1 mg of purified goat IgG was conjugated with varying amounts of SMCC-xlAPC (from 0.8 to 3.2 mg). The conjugates formed were clarified in a microfuge and the supernatants purified on a Biogel A0.5M column. For each conjugation the following were determined:
- the % of starting APC which was incorporated in soluble conjugates of IgG and APC
- the % of starting APC which was incorporated in insoluble conjugates of IgG and APC
- the % of starting APC which remained soluble and unincorporated into conjugate
- the % of total IgG-APC conjugate formed which was soluble
Note: a + b + c = 100%.
Actual results would vary to some extent from conjugation to conjugation depending on the precise concentrations of the reactants and the particular antibody or target protein. However, some general conclusions can be drawn:
- Low molar ratios of APC to target, while they give excellent incorporation of APC into conjugate, can result in yield losses through excessive cross-linking and precipitation of the conjugate formed.
- High molar ratios of APC to target result in higher amounts of unconjugated APC. These losses may be compensated for through improved yields of soluble conjugate.