Phospho-specific antibody production services is designed specific for the detection of post-translational modification. These packages includes:
- Free Antigen
- Phospho and Non-phospho peptide synthesis on serine, threonine and tyrosine residues
- Immunization of 2 host species using 70 days immunization production
- Protein A and double affinity purification on certain packages
- ELISA, SDS and Western characterization on certain packages
Antigen Design
The first step in the development of custom-made phospho-specific antibodies is the selection and design of the immunizing peptide. This is a crucial step because the specificity and utility of the custom phospho-specific antibodies depends critically on the design of the immunizing peptide. Bio-Synthesis provides design assistance at no additional charge. Please send your sequence(s) to our Sales team with any details on regions to target/avoid, and we will be happy to send our recommendations. Once the final sequence(s) has been chosen, we can then put together a formal quotation for the custom phospho-specific antibody project.
Peptide Synthesis
After sequence bee determined, two type of peptides are synthesized been synthesized at our laboratory.
- Phopshorylated Peptide: phosphorylated residues can be incorporate at Serine (S), Threonine (T), Tyrosine (Y). Cys residues been added at either N- or C-terminus for carrier conjugation with KLH or BSA. For this reason, we recommend avoiding sequences that contain internal cysteines whenever possible.
- Non phosphorylated Peptide: the peptide sequence of interest without the added phosphorylation on the residue of interest.
We have experience synthesizing peptides containing multiple phosphates; actually we have assembled peptides containing up to four phosphorylated sites; these are certainly are more challenging and there are certain guidelines that should be followed in order to enhance success.
Conjugation
Conjugate the phosphorylated peptide to KLH or BSA via thiol group of Cystein residues.
Immunization and Antibody Generation
Immunize phospho-peptide conjugate into animal(s) and collect sera at scheduled intervals .
Phospho-Specific Purification
Serum containing a mixture of phospho and non-phospho antibodies in various proportion, corresponding to the non-phosphorylated version ( formed in the animal via native phosphatase activity of the host) and also pan-specific antibodies, which can be formed against the rest of the peptide, that do not contain the phosphorylated residue.
In order to obtain to purify the desired antibody generated against the phosphorylated peptide , we first prepare an IgG fraction from the serum and apply it to our immuno-affinity purification column, a two columns purification procedure.
Step 1 Phospho peptide column: Bind the phospho peptide to gel matrix for column. This will bind only the phospho-specific antibodies and the pan-specific antibodies. The non-phosphospecific antibodies will flow through the phospho column. The phospho and pan specific antibodies can be eluted from the column together with the entire complement of additional IgGs that were isolated from the rabbit serum.
Step 2 Non-phospho peptide column: Bind the non-phospho peptide to gel matrix for purification. This column will bind the pan specific antibody only and the phospho specific antibodies does not bind to the column will be in the flow-through which contains our desired phospho-specific antibodies.
Antibody Characterization
The final stage of custom phospho-specific antibody production is the characterization of the antibodies. We use two methods:
ELISA: Phospho peptide to run an ELISA on the serum, phospho affinity purified antibody, and the flow-through resulting from the phosph column.
Western Blot: The first Western blot analysis simply demonstrates that there are no cross-reacting bands labeled by the antibody in a homogenate or cell lysate. The second level of specificity uses Western blots of phospho- and dephospho-proteins to demonstrate the selectivity of the phospho-specific antibody.