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SPOTs Frequently Asked Questions |
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Custom Peptide Synthesis |
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What is the recommended peptide length?
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What is the recommended amino acid offset?
- How much peptide is synthesized per spot?
- What is the fidelity of the synthesized peptide?
- Does secondary structure formation occur with peptides that are synthesized on the membrane?
- How does Sigma-Genosys QC the synthesis of SPOTs peptides in the custom SPOTs service?
- What is the stability and the appropriate storage conditions for the membrane?
- How many times can the membrane be regenerated and reprobed with antibody?
- Can epitope analyses be conducted with polyclonal antisera?
- Can HRP or AP conjugated secondary antibodies be used for epitope analysis?
- SPOTs DISCLAIMER
What is the recommended amino acid offset?
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| Most peptide epitopes for antibody applications are between 3-6 amino acids in length. Therefore, it is recommended that the offset does not exceed 3 amino acids in order to localize the epitope. An offset of 1 is recommended for finer mapping of the epitope. |
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How much peptide is synthesized per spot?
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A typical synthesis is expected to yield between 5-10 nmol (6-12 µg for an average 10mer peptide). The amount of peptide synthesized is determined theoretically from calculating the nmoles of free amines available for coupling per spot and assuming a coupling efficiency of >98% per cycle. |
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What is the fidelity of the synthesized peptide?
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The protocol incorporates an acetylation step at the end of each cycle to "cap" any unreacted free amines with acetic anhydride. This prevents them from coupling to any subsequent amino acids and virtually eliminates the synthesis of deletion sequences. The purity of the peptides synthesized varies for each peptide and is dependent upon sequence and length. Our analyses have determined that peptide purity is typically >70% for average 6-15 mers (by HPLC and mass spectroscopy). |
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Does secondary structure formation occur with peptides that are synthesized on the membrane?
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Yes. Secondary structures in peptides can form in sequences as short as 6 amino acids. However, the secondary structure formed by a peptide on a SPOTs membrane may not necessarily resemble the secondary structure formed by the same peptide within the full length native protein. Hence, epitope mapping results should be interpreted with the caveat that the sequence recognized by the antibody may form a conformational epitope. |
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How does Sigma-Genosys QC the synthesis of SPOTs peptides in the custom SPOTs service?
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Sigma-Genosys synthesizes three standard control peptides at the end of every custom SPOTs project, of which 2 positive and negative control peptides are probed with a standard monoclonal antibody. A third peptide is synthesized with a cleavable linker that enables cleavage of the peptide from the membrane for analysis with mass spectroscopy for mass determination and HPLC for purity. |
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What is the stability and the appropriate storage conditions for the membrane?
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The derivatized membrane is very stable prior to peptide synthesis and can be stored indefinitely when kept desicated at -20° C. Following peptide synthesis, the membrane should be stored in identical conditions for 6-8 months, however the shelf life will be significantly diminished. The stability of the peptides varies according to the sequence and is susceptible to oxidation on Met, Trp, and intramolecular bridging through Cys. |
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Can epitope analyses be conducted with polyclonal antisera?
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Yes. Although polyclonal antisera contain many antibodies that recognize a variety of epitopes on a protein or peptide, only a proportion of these sequences will be antigenic. SPOTs technology can been used to identify the predominant immunogenic sequences in a peptide or protein with polyclonal antisera. |
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Can HRP or AP conjugated secondary antibodies be used for epitope analysis?
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We do not recommend the use of HRP or AP enzymes for colorimetric detection. The reaction products of these enzymes cannot be removed effectively from the membrane when regenerated according to the regeneration protocol. However, HRP and AP enzymes can be used with chemiluminescent detection protocols for analysis of the SPOTs membrane. |
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SPOTs DISCLAIMER
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Sigma-Genosys guarantees that peptides synthesized according to the SPOTs methods detailed in Sigma-Genosys publications will result in the synthesis of linear peptides with the desired sequence and attached to a cellulose membrane through C-terminal ends. Sigma-Genosys does not make claims as to the ability of peptides synthesized on SPOTs membranes to perform in solid phase assays conducted by the researcher. |
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