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Custom polyclonal antibody/peptide combined package
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Custom polyclonal antibody/peptide combined package

Phases  (Production Protocol)
Lead Time
I. Peptide Design and Synthesis 
Custom Peptide synthesis up to 15 amino acids. Peptide with more than 15 amino acids will be charged for $15 per additional residue. We can provide assistance on designing peptide with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability with reasonable fee.
2 weeks
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 
Conjugated peptides or recombinant proteins are used to immunize rabbits. 
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 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 day
         intervals 
      •  50 ml antiserum bleed after 50 days)

7 weeks 
     VI. ELISA Test
      •  Will conjugate peptide to BSA 
         ELISA test for final total bleed

1 week
     VII. Purification
      •  Peptide specific affinity purification 
        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 adjavant 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  
  needle gauge 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). 
   First immunize 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 provided 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:
  1. 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
  2. 10 mg non-phosphospecific peptide (up to 15 amino acids) used for affinity depletion
  3. Immunization of 2 rabbits
  4. Phosphospecific and non-phosphospecific peptide ELISA titration of antibody for final bleeds
  5. 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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