Thermo Scientific Pierce Conjugate Purification Kit is a gentle affinity chromatography system for purifying horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated antibodies from unreacted enzyme after labeling.
This conjugate purification kit is designed to eliminate excess, unconjugated HRP or Alk-Phos enzyme from labeled antibodies (IgG) after performing antibody-enzyme conjugation reactions. The column of metal-chelate agarose resin binds to IgG-enzyme conjugates but allows non-conjugated enzyme (HRP or AP) to to pass through. A gentle, non-denaturing elution buffer recovers the fully active IgG-HRP or IgG-AP conjugate. The included desalting columns exchange the purified conjugate into any buffer of choice, ready for use in various immunodetection methods.
- Specific for IgG – procedure is designed for removal of HRP or AP enzymes from IgG antibodies; not for use with IgM, IgY, Fab fragments or streptavidin conjugates
- Complete kit – includes affinity column, activator and elution buffers, desalting column and storage buffers for HRP and AP conjugates
- High capacity – the single-use column-kit is designed for purification of 0.5 to 50mg of antibody conjugate dissolved in Tris-buffered saline
- Improves assays – removing unconjugated enzyme from antibody conjugates often reduces background and improves overal performance of immunoassays
In this kit procedure, the nickel-chelated agarose binds IgG through a histidine-rich cluster on the Fc region at the junctures of the Cγ2 and Cγ3 domains, which is highly conserved across all mammalian IgGs. Purified IgG from sheep, mouse, goat, rat and rabbit will bind to nickel-chelated resin. Avidin or streptavidin enzyme conjugates cannot be purified by this method because they do not bind to the nickel-chelated agarose. Additionally, this kit cannot be used to purify antibodies from serum because other serum proteins will also bind to the nickel column.
Removing non-conjugated enzyme using this kit can improve assay results. Increase in assay sensitivity, shorter washes and incubation times, and a high signal-to-noise ratio can be more easily achieved when free enzyme is absent from the system. Furthermore, the column concentrates samples, which eases assay optimization when using conjugates in dilute solutions.
|Purification of IgG-HRP removes excess non-conjugated enzyme and recovers fully active HRP conjugated antibody. The solution resulting from a somewhat inefficient conjugation reaction (5mg total protein) was loaded to the nickel-charged column and purified using the Conjugate Purification Kit. Test samples from various time-points in the procedure were tested for HRP activity by in a microplate assay using ABTS (Part No. 37615) as a substrate.
- Boorsma, D.M. and Kalsbeek, G.I. (1976). A comparative study of horseradish peroxidase conjugates prepared with a one-step and two-step method. J. Histochem. 23, 200-207.