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Custom phospho-specific polyclonal antibody service
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Custom phospho-specific polyclonal antibody service

Phases
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:
Subject:
  New Zealand White Rabbits
Adjuvant:
  Complete Freund's Adjuvant (CFA) initially, followed by incomplete
Freund's Adjuvant (IFA) for all subsequent injections.
Immunogen:
  Typically, proteins and conjugated peptides. Amount of immunogen used
for each immunization is 0.5 mg /per rabbit /per injection.
Procedure:
  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.
Bleeds:
  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.
 
Schedule:
 
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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