What peptide purity and design requirements do I need for phospho specific antibodies?
Phospho-specific antibody production against a peptide antigen containing a phospho-residue is a bit more complicated than traditional peptide antibody production. Animals can produce antibodies against additional epitopes in the peptide sequence as well as produce antibodies that do not contain the modified phospho-residue. Since a pool of phospho and non-phospho antibodies can be difficult to work with Bio-Synthesis offers highly purified phospho-peptides and phospho-specific antibodies to minimize this type of problems. The selective purification of phospho-peptide specific antibodies from anti-serum allows the removal of the non-phospho antibodies from the polyclonal antibody pool. The availability of both, phospho-specific and non-phospho-specific antibodies can be very helpful for studying protein pathways.
Factors to consider:
- Length of the peptide used for antibody production:
Use a peptide with a length of 12 amino acid residues or less. The length of the peptide is important due to the number of epitopes available to the host animal’s immune system. The minimum length of an epitope is 6-8 amino acids. Using a sequence with 12 or fewer residues ensures that the phosphorylated residue will be included in all antibodies produced.
- Position of Phosphorylated Residue:
Place the phosphorylated residue in the middle of the peptide sequence. This increases the probability that all antibodies produced against the sequence will have epitopes including the phosphorylated residue.
Use a phospho peptide with >90% purity. However, the purity for the non-phosphopeptide can be >85%. We highly recommend to our clients to select our superclass phospho-specific peptide package to ensure that the antibodies are produced against the exact sequence of interest. This also applies any peptide modifications which require two columns affinity specific purification (e.g. acetylated lysine).
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, two step purification process is been used in our laboratory which is designed to detect the presence of modified amino acid in a sequence specific context.
Antibody Purification using Tandem Affinity Chromatography
Bind or conjugate the phospho peptide to a gel matrix to be used in the first column. This will allow to affinity capture 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.
Conjugate the non-phospho peptide to the gel matrix use in the second column for the purification. This column will affinty capture or bind the pan specific antibodies only. The phospho specific antibodies that do not bind to the column will be in the flow-through which contains the desired now highly specific 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 phospho column. This process is included in our custom phospho-specific antibody packages: FIIP-PH-RA120I, SIIP-PH-RA120I
- 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. This process included in our custom phospho-specific antibody packages: SIIW-PH-RA120I
All these packages isolate both phospho-specific antibodies and the non-phospho antibodies for your protein. The corresponding antibodies can be used separately or in conjunction to study your protein’s regulatory pathways.
Please contact BSI for more info.