Custom Phosphospecific Antibodies - $2,975 for a
single phosphorylation site antibody (multiple project
discounts available - contact us for current specials)
all inclusive NO hidden charges!
Dual phosphospecific antibodies also available
Peptide Sequencing by CID MS/MS included at no extra charge!
As part of our innovative PhosphoExplorationTM Program, 21st Century Biochemicals offers the production of phosphorylation state-specific polyclonal antibodies. Our scientists have many years of experience manufacturing phoshospecific antibodies that recognize only the activated (i.e., phosphorylated) form of the protein target. Key to the manufacture of a scientifically relevant phosphospecific antibody is the assurance that it reacts with the proper phosphorylated protein in its proper form. In brief this means that if the site of interest contains two nearby amino acids that must be phosphorylated in order for the protein to be in its 'activated' state (i.e., MAP kinases), then the antibody being developed to detect this 'activated' state must recognize the dually phosphorylated protein and only the protein in this form. Oftentimes, this might require the manufacture of both monophosphorylated peptides as well as the nonphosphopeptide for affinity depletion of the serum. In addition, many phosphorylation sites are related, with a standard sequence motif being required for recognition and phosphorylation by the specific kinase involved. Thus, homology analysis of the amino acids flanking the phosphorylation site is also important to insure that the antibody produced will be specific for the intended phosphoprotein.
In addition to expert technical support for each antibody project, 21st Century Biochemicals is also the only company to offer MS Check™ peptide sequence confirmation for all phosphospecific antibody protocols at no extra charge! The sequence of each peptide used for phosphospecific antibody production is confirmed prior to immunization, thus ensuring that the animals are immunized with the correct peptide. This level of quality control is unsurpassed in the industry and is available only from 21st Century Biochemicals.
Our complete phosphospecific antibody protocols include:
- consultation with our scientific staff
- production of a purified phosphopeptide and the corresponding
nonphosphopeptide; 2-5mg of the phosphopeptide and the nonphosphopeptide sent to the researcher
- preimmune serum from each rabbit (two)
- Conjugation of the peptide to our proprietary carrier for immunization and to BSA for ELISA analysis; ELISA of bleed 4 versus both the phospho and nonphosphopeptides
- 5 immunizations and 4 production bleeds per animal, 95 day protocol
- manufacture of an affinity depletion column using the nonphosphopeptide
- manufacture of an affinity purification column with the phosphopeptide
- serum samples sent to customer for evaluation
- affinity depletion and affinity purification of 50ml of serum
- affinity purified phosphospecific antibody sent to customer
- the animals are kept alive for 2 weeks while you evaluate your serum rather than immediately sacrificing them; more serum and/or purified antibody can be ordered at this time
The above is the typical scenario for a phosphospecific antibody designed to detect a single phosphorylated amino acid. In the situation where two or more residues are phosphorylated and they are close enough to permit the manufacture of a single antibody for dual phosphor detection, the costs would be higher. Generally, the amino acids should be within 5 residues of each other to be included within the same epitope. The further away the two phosphorylated amino acid are from one another, the lower the chances for successfully manufacturing one antibody to recognize both phosphorylated sites. In this case, it may be possible to manufacture two antibodies that would each recognize one of the phosphorylated residues but not the other (nonoverlapping antibodies).