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Purification Information
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SERUM AFFINITY PURIFICATION OPTIONS

Affinity purification with immobilized Protein A, G, A/G: Depending on the animal type or request, a project specific Protein A, Protein G, or Protein A/G column is prepared to purify the quantity of serum indicated.

Affinity purification with immobilized antigen: Antigen-affinity purification includes preparation of one purification column with the peptide/protein covalently attached to purify the antibodies specific for the antigen. Antigen-affinity purification also includes an ELISA analysis of the purified antibody and flow-through (see below for more detail).

Affinity purification with immobilized antigen and negative adsorption: Antigen-affinity purification with negative adsorption includes the preparation of two purification columns to isolate the specific subpopulation to a modified peptide (positive selection) versus the unmodified peptide (negative selection). The serum is first purified with the modified peptide column (e.g. phospho-specific peptide) and then passed over a control column (e.g. non-phosphorylated peptide). The flow-through from the control column will contain the antibody specific to the modified protein only, while the eluent from the control column contains the cross-reactive antibody that binds to both the modified and unmodified forms of the protein. ELISA analysis of these purified antibody subpopulations and flow-through is included.

Affinity purification of Chicken IgY: IgY purification is available for the purification of up to nine egg yolks, which require an alternative purification method to Protein A or Protein G. The Thermo Scientific Pierce Chicken IgY Purification Kit uses a delipidation-and-precipitation procedure that is optimized to recover approximately 100mg 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, because it is dissolved in PBS and thoroughly clarified, it can be further affinity-purified against a specific antigen,.

ANTIGEN-AFFINITY PURIFICATION DATA

Purified antibody samples from antigen-affinity purification are titered by indirect ELISA and reported to the customer to evaluate and quality-assure the purification protocol. Titers from the purified antibody (the eluent) and the remaining serum (flow-through) are measured against the solid-phase bound peptide or protein. The titer from the eluent indicates the minimum concentration at which the purified antibody can effectively detect the antigen, while the flow-through titer represents the reactivity of the antibodies remaining in the depleted serum. The antibody titer value (given in ng/mL) indicates the concentration of the antibody where detection of the coated peptide is above background. The flow-through titer is determined by taking the serum that passed through the column and performing 1: 1 serial dilutions with the neat serum across the plate. (Note: the flow-through is reported as the reciprocal of the dilution as the remaining antibody concentration cannot be determined since this is a mixture of proteins.) The affinity purified antibody titers are reported differently than crude serum titers because the exact amount of specific anti-peptide or anti-protein antibody in the solution is known. The eluent titer is reported as a concentration, which enables the reporting of much more quantitative values versus the qualitative dilution value with crude serum. If your antibodies have been negatively adsorbed against any related protein or peptide, such as the non-phosphorylated analog peptide, these results will also be reported to show the preference for the modified versus unmodified forms. If your antibodies were made against multiple peptides (co-inject project) the titer results will be listed against both peptides.

ADDITIONAL PURIFICATION NOTES

Various immobilization procedures are available for the purification process through our custom services using our SulfoLink, AminoLink, CarboxyLink, or GlycoLink Kits and resins. For a detailed overview of affinity purification, please review our Affinity Purification Overview page.

If you need assistance in determining the right purification method, please contact us by phone at 1-800-874-3723 X307 or e-mail at abs@thermofisher.com.

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