Molecule guides
Peptide Antibodies and Antisera: Formats, Immunogens and Validation Controls
What you are actually buying when a listing says "antibody", why the immunogen is the real specification, and which controls make a result defensible.
Detection in peptide research runs on polyclonal antibodies and antisera: raised in a host animal against a defined peptide immunogen, they pick out the target in tissue sections, on blots and within immunoassays. Polyclonals outnumber monoclonals in the antibodies section of the catalogue for a concrete reason: a short peptide offers only a handful of epitopes, and a polyclonal response spanning several of them finds a molecule of perhaps ten residues far more dependably than one monoclonal clone can.
Three formats behind the word "antibody"
Our glossary entries on antiserum, purified IgG and polyclonal versus monoclonal explain the distinctions at greater length. Each catalogue listing names the format it supplies, because "antibody" on its own does not specify a reagent.
Why the immunogen is the specification
How a peptide antibody behaves is determined entirely by what it was raised against. The immunogen sequence identifies the region of the parent molecule that is recognised, and that settles three things worth knowing before you order. Can it separate processed forms? An antibody made against a peptide's C-terminus will miss a C-terminally extended precursor, which is exactly right when the mature hormone is the target and exactly wrong when total content is. Will it cross-react inside the family? Antisera directed at the conserved mid-region of proglucagon products bind glucagon, glicentin and oxyntomodulin equally. Does it demand a modification? Phospho-specific and amidation-specific reagents come from modified immunogens and should ignore the unmodified sequence, and that selectivity is the whole point of them in tau and neuropeptide studies.
Host species and species reactivity are not the same thing
These two specifications are distinct and both count. Host species, normally rabbit and sometimes goat, sheep or guinea pig, dictates the secondary antibody required and which primaries can share a multiplex. Two rabbit primaries cannot be told apart by secondary on one section. Species reactivity describes which organisms' version of the target is recognised, and it depends on sequence conservation rather than on the host. A rabbit antibody raised against human amyloid-beta may or may not bind the rodent sequence; the listing gives tested reactivity, and anything untested should count as unknown rather than assumed. See species reactivity.
Controls that make a result publishable
Specificity of antibodies is the single biggest reproducibility problem in peptide research, and the required controls are long established. Chief among them is the blocking peptide: pre-incubate the antibody with an excess of immunogen peptide, and genuine staining or banding should vanish while non-specific signal stays. Most antisera in the catalogue have a matching blocking peptide available, and running it separates a suggestive picture from evidence. After that come a knockout or knockdown sample where one exists, a secondary-only control and, for anything quantitative, independent confirmation by mass spectrometry or by a second antibody against another epitope. In fields where antibody validation has been publicly challenged, irisin being the obvious case, these controls are simply mandatory.
Reading the application specifications
Listings give validated applications with suggested working dilutions, usually immunohistochemistry, immunofluorescence, Western blot, ELISA, RIA and immunoprecipitation. Two warnings follow. An antibody validated in one application may not work in another: reagents that read conformational epitopes frequently perform in immunohistochemistry and fail on a denaturing Western, while linear-epitope reagents often behave the other way round. And suggested dilutions are starting points for titration in your own system rather than fixed numbers, since sample type, fixation and detection chemistry all move the optimum. Immunoprecipitation normally pairs the antibody with magnetic beads, while labelled antibodies supply the conjugates for direct detection.
Storage and handling
Because antibodies are proteins in solution, they are handled quite unlike lyophilised peptides. Aliquot them as soon as they arrive; this matters more here than for virtually anything else in the catalogue, since each freeze-thaw cycle aggregates IgG and permanently cuts titre. Keep single-use aliquots at −20 °C, or hold a working aliquot at 2-8 °C for short-term use, and watch the preservative named on the vial: sodium azide is common and incompatible with horseradish-peroxidase detection, so azide-containing antibodies need dialysis before HRP conjugation. Reconstitute lyophilised antisera in the stated water volume and aliquot them straight away. Steer clear of frost-free freezers, whose defrost cycles repeatedly part-thaw the stock. General principles are in the aliquoting guide and the storage guide.
How antibodies relate to the other product types
Choose an antibody when the endogenous molecule needs detecting or localising in tissue, cells or a lysate. Add the matching peptide standard to serve as the blocking-peptide control and as a positive control on a blot. Choose an ELISA or RIA kit when the question is how much target a sample contains, since a kit packages a matched antibody pair, standards and a validated range, and is quicker and more reproducible than building the assay from parts. Begin at the catalogue hub, or at a topic such as Alzheimer's, cancer or diabetes, where antibody choice bears most of the experimental load. Every catalogue item is supplied for in-vitro and approved animal research only, and none is a diagnostic reagent.
Questions
Why are antibodies against peptides normally polyclonal?
Short peptides display very few epitopes. A polyclonal preparation covers several at once, so detection survives when one epitope is hidden by fixation, denaturation or a bound partner. Monoclonals bring lot-to-lot consistency and single-epitope precision, valuable in phospho-specific work, but for most small peptides a well-purified polyclonal finds the target more reliably.
Whole antiserum or affinity-purified antibody?
Antiserum suits RIA and EIA, where some background is acceptable and cost per assay counts. Affinity-purified material suits immunohistochemistry, immunofluorescence and low-abundance Westerns, where non-specific serum immunoglobulin creates background no blocking step will clear. Protein A/G purified IgG falls between them and earns its place mainly when a defined IgG concentration is needed.
How is specificity demonstrated?
Run the blocking-peptide control: pre-incubate with excess immunogen peptide and show the specific signal disappearing while background remains. Include a secondary-only control and, where obtainable, a knockout or knockdown sample. Quantitative claims should be backed by a second antibody against a different epitope or by mass spectrometry, and the controls belong in your methods.
Can one antibody cover both immunohistochemistry and Western blotting?
Only when both have been validated. Reagents binding conformational epitopes often succeed on fixed tissue yet fail after SDS denaturation, and linear-epitope reagents commonly do the opposite. Listings show tested applications, and an application absent from that list is untested rather than merely discouraged, so validate it yourself before relying on it.
Why does antibody performance fall off over time?
Freeze-thaw, nearly always. Every cycle aggregates part of the IgG, and those losses accumulate irreversibly. Divide the stock into single-use volumes on arrival and never refreeze a working aliquot. Frost-free freezers aggravate the problem through their defrost cycles. An aliquot in active use is best kept at 2-8 °C with the stated preservative.
What does sodium azide in the vial affect?
It keeps the antibody preserved but inhibits horseradish peroxidase, so an azide-containing reagent cannot be conjugated to HRP or used in certain direct-detection formats without dialysis first. Azide is also toxic and needs proper disposal. Azide-free presentations exist for conjugation work, so check the buffer composition given in the listing.