Custom Polyclonal Antibody Purification

Antigen-specific antibody purification is the method of choice for most researchers when antibodies have been generated against peptides. This process enables maximum isolation and enrichment of only the most specific antibodies generated to the antigen while removing other unwanted pre-immune antibodies. Many applications, such as IHC and IF, benefit from this process as antibodies can be used at higher concentrations (lower dilutions) without increasing signal background.

Once antiserum has been selected for purification, it is pooled and the IgG fraction of antibody is isolated by ammonium sulfate precipitation. This removes many of the sticky proteins from the antiserum and allows for a cleaner preparation of material to be subjected to the affinity column. Affinity resins are prepared by cross-linking antigens (primarily peptides) by the appropriate amines, acids, or sulfhydryls dependent upon the carrier conjugation chemistry used to generate the immunogen. After antiserum is incubated in the column, antibodies are eluted using step-wise pH gradient and collected in neutralizing buffer. Purified antibodies are then concentrated and final concentration is measured by BCA. Antibodies are titered by ELISA and reported to the customer. Both eluent and flow-through titer is reported. We employ several key procedures to provide the best possible affinity purified antibodies. These procedures not only help ensure better specificity but also help maintain maximum antibody utility and minimize loss of desired binders.

• Antibodies are isolated and exposed to depletion columns to remove unwanted binders and enrich for target antibodies.
• pH elution gradient is used to capture diverse polyclonal species and minimize antibody pH lability.
• Affinity ligands are bound to matrices through multiple attachment points and mimic antigen presentation in vivo, increasing capturing efficiency.

Our multi-step gradient elution process minimizes irreversible denaturation of pH susceptible antibodies. This is extremely important because polyclonal antibodies vary in affinity and general characteristics and many populations behave differently. For more detailed information, email or call to speak with one of our project specialists.

Antibodies to post-translational modifications, such as phosphorylation or acetylation, benefit from the use of a multi-column positive and negative purification protocol. Our monospecific antibody approach includes the use of up to three columns to enrich for the modification while depleting away antibodies that may cross-react between the modified and unmodified protein.

Phosphospecific antibody example

Antiserum is first incubated with the phosphopeptide column and then antibodies are eluted and subjected to a column containing the non-phosphopeptide. The flow-through is captured from this column and concentrated to make the final product. Antibodies that stick to the non-phosphopeptide column are also eluted and both antibody fractions are titered against the phosphopeptide and non-phosphopeptide to characterize the reactivity against the modification and to provide a degree of preference for the modification. A complete titer report is supplied to the customer after shipment of the final purified antibodies.

Protein A/G purification is the purification method of choice for polyclonal antibodies that have been generated against recombinant proteins or antigens that are not available in sufficient quantities to make antigen affinity columns.

Polyclonal antiserum is selected by the customer from the best performing animal or from pooled animals and subjected to our Pierce Protein A or G resin using the appropriate volume based calculations to help ensure complete capture of all IgG antibodies. After incubation, antibodies are eluted from the column using 0.1M glycine (pH 2.0) and neutralized in 1M phosphate.

Typical IgG affinity resins (Protein A or Protein G) do not bind chicken IgY and therefore are not effective for IgY purification. The Pierce Chicken IgY Purification Kit uses a delipidation-and-precipitation procedure that is optimized to recover approximately 100 mg of 90% pure IgY from fresh egg yolks of appropriately immunized chickens. The resulting polyclonal IgY is sufficiently pure for direct use in many immunodetection procedures, or it can be further affinity-purified against a specific antigen, because it is dissolved in PBS and thoroughly clarified.

After a customer confirms the specific reactivity of antibody in corresponding chicken antiserum, eggs collected are pooled and delipidation reagent and stirred and incubated for 2-24 hours at 4°C. Clear supernatant is decanted and added to IgY precipitation reagent and further incubated for 1-2 hours. The solution is then centrifuged and the resulting pellet brought back into a PBS solution and OD measured. Subsequent affinity purification is now pursued.

We also provide integrated services for chemically labeling purified monoclonal antibodies to create conjugated antibodies for use in nearly any assay system. Let us know your needs and we can include this option when we provide a project quotation.