Phospho-Specific Polyclonal Antibody | MédiMabs

Phospho-Specific Polyclonal Antibody

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Phospho-Specific Rabbit Polyclonal Antibodies

 

Phospho-specific antibodies are affinity-purified antibodies with specificity to a target sequence that is phosphorylated on a determined tyrosine (Y), threonine (T) or serine (S) residue. Phosphorylation is a post-translational modification of proteins involved in many cellular processes, making their detection crucial for the study of regulatory mechanism ranging from cell division in cancer to neuronal signal transduction in learning and memory.

 

The design of the peptide to be used as antigen is the pivotal step for the production of phospho-specific antibodies. MédiMabs will advise you on:

  • The length of the peptide to be used
  • The position of Phosphorylated Residue
  • Antibody Purification

 

Phospho-specific antibodies must be isolated from the non-phospho-specific and pan-specific antibodies by way of a two-step purification process:

 

Step 1: The whole polyclonal serum is applied to the phosphorylated peptide column. In this step, the non-phospho-specific antibodies will not bind to the column and will stay in the flow-through together with the entire complement of additional IgGs that were isolated from the rabbit serum. The phospho-specific antibody and the pan-specific antibodies will bind to the column and are then eluted from the phospho-peptide affinity column.

 

Step 2: The elute from step 1 is applied to the non-phosphorylated peptide column. In this step, the pan-specific antibodies and the non-phospho-specific antibodies (if some remain) will bind to the column. Phospho-specific antibodies will pass through and will be recovered in the flow-through.

 

MédiMabs standard protocol for production of phospho-specific antibodies in rabbit

 

130-Days Protocol:

Antigen design and synthesis following precise criteria to maximize success. The synthesis of the peptide includes:

  • Phosphorylated peptide coupled to KLH for immunization
  • Phosphorylated peptide coupled to BSA for antibody evaluation
  • Non-phosphorylated peptide coupled to BSA for antibody evaluation
  • Non-coupled phosphorylated peptide for purification
  • Non-coupled, non-phosphorylated peptide for purification
  • A minimum of 90% purity for each peptide

The production of the polyclonal serum includes:

  • Control serum collection
  • 6 Immunizations of two Rabbits
  • Test bleeding
  • Titer evaluation by ELISA
  • 2 Boost immunizations
  • Production bleeding
  • Final bleeding

The purification of the phospho-specific antibody includes:

  • Double-step affinity purification of 20 ml of polyclonal serum
  • Antibody evaluation by ELISA

Deliverables:

  • 5-10 ml of pre-immune serum of each rabbit
  • 70-100 ml of polyclonal antibody per rabbit
  • Purified antibody derived from 20 ml of serum (a minimum of 1 mg of phospho-specific antibody). Purification of supplementary serum available, please inquire.

Rabbit is the most commonly used animal to raise polyclonal antibodies. Improvements in the rabbits' health through better nutrition and sanitation has made the raising and maintenance of rabbit populations easier, making them increasingly useful as a research tool.  New Zealand white rabbits are a good model because this animal is small enough to be easily manipulated but big enough to provide 20 mL/week of blood (and a total of 150-200 mL during a standard project (2 Rabbits).

 

The final volume of serum obtained during a standard project (2 rabbits) is between 140 and 200 mL and can be used as is for multiple immunoassays. 

 

40 mL  blood (in 2 weeks) = 200 mg IgG including 5% specific (10 mg)

 

For more sensitive techniques, standard method of IgG purification is a fast and efficient way to improve the performances of the antibodies. If even more specificity is neccesary, affinity purification is also offered to select for the most specific antibodies with highest affinity.

 

Polyclonal antibody generation in Rabbits is one of the fastest and the most inexpensive way to get good quality antibodies

 

Advantages of rabbit polyclonal antibodies :

 

Affinity: the average affinity of monoclonal antibodies is generally lower than that of polyclonal antibodies

Effector Functions: monoclonal antibodies may not produce the desired biologic response

Specificity: monoclonals against conformational epitopes on native proteins may lose reactivity with antigens that have been minimally perturbed

Delivery time : a custom polyclonal antibody can be delivered in less than 3 months

Cost : MédiMabs is in the position to offer top quality polyclonal antibodies at a more affordable price than most suppliers on the North American market