Our Custom Phosphospecific Antibody Production package includes everything needed to obtain affinity-purified antibodies that are specific to a phosphorylated epitope. We run an extensive series of ELISA assays on the production bleeds and on the affinity purified antibodies to ensure that antibodies are specific to the phosphorylated peptide and show no activity against the non-phosphorylated peptide.

Service Details Price
Package #4: Rabbits
  • Protein Sequence Analysis
  • Synthesis of up to a 15aa Non-Phosphorylated Peptide
  • Synthesis of up to a 15aa Phosphorylated Peptide
  • Conjugation of 3-5 mg of the Phosphorylated peptide to KLH
  • 13-week Antibody Production Protocol:
    • 2 Rabbits
    • Pre-Immune Serum from each animal
    • 4 Immunizations per animal
    • 4 Production Bleeds of 20-25 ml of serum per animal
    • Provides approximately 160-200 ml of anti-sera
  • ELISA on the 1st Production Bleed against both peptides
  • Affinity Purification of 25 ml of serum against the non-phosphorylated peptide
  • Affinity Purification of 25 ml of serum against the phosphorylated peptide
  • Full ELISA verification of purified antibody specificity
  • Both re-useable columns included
  • Handling / Shipment Preparation
  • $150.00 Package Pricing Discount included
Service Details Price
Peptide Cost per each additional peptide residue over the 15 residues included with the package $20.00
4-Week Extension Provides 1 immunization and 2 additional production bleeds per animal. Provides approximately 80-100 ml of serum. (Covers both animals) $245.00
Exsanguination Bleeds Terminal Bleeds that provide approximately 50+ ml of serum per animal. (Covers both animals) $120.00
200 ml Affinity Purification Upgrade of each 25 ml Affinity Purification to 200 ml Affinity Purifications. Larger columns included. $800.00
Sodium Azide Preservative for antiserum No Charge

Antigen Design:
Pacific Immunology® includes antigen design assistance with this package service. We use our innovative and proprietary combination of algorithms to analyze your protein sequence and predict which regions are immunogenic, unique and exposed.

Conjugation of Peptide to Carrier Protein:
Because peptides are too small to generate an immune response by themselves, conjugation to a carrier protein will be required for immunizations. Pacific Immunology® recommends coupling peptides to KLH (Keyhole Limpet Hemocyanin).

Terminal Cysteine:
Pacific Immunology® recommends coupling the phospho peptide to the carrier protein via a cysteine that has been added to the N or C-terminal of the peptide. For this reason, we recommend avoiding sequences that contain internal cysteines whenever possible.

Quantity of Peptide:
Pacific Immunology® typically synthesizes 40-50 mg of immunograde peptide, with approximately 5-10 mg of each peptide being required for conjugation to the carrier protein, ELISA and affinity purifications. All unused peptide will be delivered to the customer.

Affinity Column:
Both the phosphorylated and non-phosphorylated affinity columns are included so that the client can run additional purifications as needed. Longevity of the columns varies, but best yields are typically obtained within 6-12 months of the project’s completion.

Titer Guarantees:
Pacific Immunology® offers one of the highest titer guarantees in the industry, promising at least a 1:100,000 antibody titer against the peptide sequence. For additional details, please read our Guarantee page in our Resources section.

Phospho Purification Strategy:
To isolate antibodies specific to the phospho epitope, serum will first be affinity purified against the non-phosphorylated peptide prior to being run through the phosphorylated peptide column. Comprehensive ELISA testing against both peptides confirms the specificity of the purified antibodies.

Purified Antibody Yields:
On average, 2-3 mg of peptide specific antibody will be isolated from affinity purification of 25 ml of antiserum. However, because antibodies specific to the phospho site will represent only a portion of antibodies against the phospho peptide sequence, the yield of phosphospecific antibodies will typically be around 0.5 – 1 mg.

General Purification Strategy:
Unless one animal has particularly high antibody titers against the peptide, we recommend affinity-purifying a pool of 25 ml serum mixed from both animals to ensure maximum epitope variability.