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Bio-Synthesis offers synthesis of peptides containing the phosphoryl (PO3) group on serine, threonine, and tyrosine residues while providing the generation of phospho-specific antibody, aimed to detect the presence of modified amino acids in a sequence specific context. These custom phospho-specific antibodies can be used to ensure that the peptide mimics the natural conformation and activity found in the protein. It is a powerful tool for protein pathway studies to detect whether a protein is phosphorylated at a particular site, or binding to and detecting phosphorylation-induced conformational changes in the protein.
Other modification specific antibodies such as methylation and acetylation are also available upon request. All these post-translational modification specific antibodies are double purified at Bio-Synthesis against the sequence and the modified peptide to ensure specificity.
BSI supplies a Polyclonal Antibody Information Sheet with each inquiry to assist in meeting customer specifications.
Select your desired Custom Phospho-Specific Polyclonal Antibody Services using Bio-Synthesis’s all inclusive production packages that are scientifically proven and have species-specific schedules. Should you not find a package that suits your needs, you have the option to develop a customized host species and immunization schedule to meet your protein detection needs.
Request a quote or contact us for your Phospho-Specific Polyclonal Antibody packages.
Peptide Phospho-specific Antibody (Rabbit) - First Class Combo
Peptide Phospho-specific Antibody (Rabbit) - Super Class Combo (Most Popular)
Peptide Phospho-specific Antibody (Rabbit) - Super Class Combo W (Western Characterized)
Peptide Phospho-specific Antibody (Rabbit) - Super Class Triplet Purification
Phospho-specific antibody production services is specifically designed for the detection of post-translational modification. These packages includes:
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 critically depends 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.
After the sequence has been determined, two types of peptides are synthesized at our laboratory.
We have experience synthesizing peptides containing multiple phosphates. Actually, we have assembled peptides containing up to four phosphorylated sites, which are certainly more challenging and there are certain guidelines that should be followed in order to enhance success.
Conjugate the phosphorylated peptide to KLH or BSA via thiol group of Cysteine residues.
Immunization and Antibody Generation
Immunize phospho-peptide conjugate into animal(s) and collect sera at scheduled intervals.
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 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 column purification procedure.
Step 1 Phosphopeptide Column: Bind the phosphopeptide 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-phosphopeptide Column: Bind the non-phosphopeptide to gel matrix for purification. This column will bind the pan specific antibody only, and the phospho specific antibodies not bound to the column will be in the flow-through, which contains our desired phospho-specific antibodies.
The final stage of custom phospho-specific antibody production is the characterization of the antibodies. We use two methods:
ELISA: Phosphopeptide to run an ELISA on the serum, phospho affinity purified antibody, and the flow-through resulting from the phospho 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.
We offer four phospho-specific antibody packages. User-design production protocol also available and can also be modified from existing package.
Continuation of your product past our standard immunization schedule is offered on a month to month basis. The standard extension protocol includes
one boosters, two serum collections, and maintenance for one month. Terminal bleeds may be obtained at any time yielding approximately ~75 ml/rabbit of additional serum taken upon customer request.