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Custom phospho-specific polyclonal antibody service

Custom phospho-specific polyclonal antibody service

Lead Time
I. Peptide synthesis
  • Peptide synthesis up to 15 amino acids.
  • Each phospho-residue (Phospho-Ser, Tyr or Thr)
2 week
II. Conjugation
  • Conjugation of 5 mg peptide (using Cysteine) to KLH.
  • 10 mg free peptide will be provided for client
1 week
III. Immunization and anti-sera production
  • Ten-week protocol to produce antibodies in 2 rabbits.
  • Pre-immune 5 ml/per rabbit and total final bleeds from two rabbits (100 ml antiserum) will be provided to client.
10 weeks
Total Lead Time
13 weeks
Optional Services:
IV. Additional MAX Bleed
      •   MAX bleed 50 ml from each rabbit

1 week
V. Extension
(two additional booster injection, 20 days interval, 50 ml antiserum bleed after 50 days)

7 weeks
VI. Test
ELISA test for final total bleed (including conjugating peptide to BSA)

1 week
VII. Purification
  • Synthesis the control peptide of the same sequence without phospho residue.
  • Phospho-peptide antibody purification by 2-steps peptide affinity purification including phospho-peptide column and non-phospho-peptide column
  • ELISA data will be included for free

1 week

Polyclonal Antibody Production Protocol
Protocol Definition:
  New Zealand White Rabbits
  Complete Freund's Adjuvant (CFA) initially, followed by incomplete
Freund's Adjuvant (IFA) for all subsequent injections.
  Typically, proteins and conjugated peptides. Amount of immunogen used
for each immunization is 0.5 mg /per rabbit /per injection.
  The immunogen is diluted to one mL with sterile saline and combined
with one mL of the appropriate adjuvant. The antigen and adjuvant are
mixed thoroughly to form a stable emulsion which is injected
subcutaneously and provides enhanced immune response from the
sustained presence of the immunogen.
  Blood is collected from the central ear artery with a 19 gauge needle and
allowed to clot and retract at 37°C overnight. The clotted blood is then
refrigerated for 24 hours before the serum is decanted and clarified by
centrifugation at 2500 rpm for 20 minutes.
Day 1
Bleed 25 mL (yields 10 mL pre-immune serum). 1 st Immunize with antigen in CFA.
Day 20
2 nd Immunize with antigen in IFA.
Day 40
3 rd Immunize with antigen in IFA.
Day 50
Test Bleed 10 mL for ELISA screen(internal quality control only, not provide to client).
Day 60
4 th Immunize with antigen in IFA.
Day 70
Final Total Bleed 50 mL from Each Rabbit.
Phosphospecific Antibody Affinity Purification:
Genemed Synthesis offers phosphospecific antibody affinity purification which includes:
  • Phosphospecific peptide synthesis (up to 15 amino acids) -- 10 mg used for phosphospecific epitope affinity purification, 10 mg used for antibody production, and 10 mg delivered to client
  • 10 mg non-phosphospecific peptide (up to 15 amino acids) used for affinity depletion
  • Immunization of 2 rabbits
  • Phosphospecific and non-phosphospecific peptide ELISA titration of antibody for final bleeds
  • Pre-immune sample of serum (5 mL/each rabbit) and anti-serum (100 mL total) from two rabbits
  • Affinity purification of serum
  • Shipment of purified phosphospecific antibody which does not cross-react with
    non-phosphospecific peptides (confirmed by ELISA)

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