Anti-Cystatin-SA Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Cystatin-SA on Western blot?

Mature Cystatin-SA migrates at approximately 14–16 kDa on reducing SDS-PAGE, corresponding to the 141-amino-acid secreted form. The CST2 gene encodes a 141-residue mature protein after signal peptide cleavage. Some groups report slight gel mobility shifts depending on glycosylation status or post-translational modifications, though Cystatin-SA lacks canonical N-glycosylation sites. If you observe higher-molecular-weight species, consider dimers or complexes with target proteases, which can survive sample preparation. Always include a saliva or secretory-fluid lysate as a positive control to confirm the expected band.

What is the recommended starting dilution for Cystatin-SA antibody in Western blot?

We recommend starting at 1:1000 dilution in blocking buffer for Western blot, based on validation with human saliva and epithelial cell lysates. This polyclonal was raised in rabbit and typically yields clean signal at this dilution with standard chemiluminescent detection. For low-abundance samples or serum, you may need to concentrate your input or increase antibody concentration to 1:500. Conversely, saliva and other secretory fluids express Cystatin-SA at high levels, so 1:2000 may suffice. Optimize empirically by running a dilution series with your specific lysate and detection system.

Does this Cystatin-SA antibody cross-react with other cystatin family members?

The cystatin superfamily includes multiple closely related members, including Cystatin-C, Cystatin-S, and Cystatin-SN, which share significant sequence homology in the conserved cystatin fold. This polyclonal was raised against human Cystatin-SA, and specificity was validated by Western blot against recombinant protein and saliva lysates. However, polyclonal antibodies can exhibit cross-reactivity with related isoforms. If your experimental system co-expresses other type-2 cystatins, run parallel blots with lysates known to contain only one isoform, or perform peptide competition assays to confirm specificity. We have not systematically tested cross-reactivity against all family members.

What sample types are best for detecting endogenous Cystatin-SA?

Cystatin-SA is predominantly expressed in saliva, tears, and other secretory fluids, where concentrations can reach micrograms per milliliter. Human saliva is the gold-standard positive control and requires minimal processing—clarify by centrifugation and load 1–5 micrograms total protein. Esophageal, oral, and upper airway epithelial cells also express CST2 at detectable levels. For cancer studies, esophageal squamous cell carcinoma and non-small cell lung cancer lines have shown altered expression. Serum and plasma contain low to undetectable Cystatin-SA under normal conditions. Always compare to a secretory-fluid control to establish baseline signal.

Is this antibody validated for use in mouse or rat samples?

This antibody is validated for human Cystatin-SA. Mouse and rat orthologues share approximately seventy to eighty percent amino acid identity with human CST2, and cross-reactivity is possible but not guaranteed. If you plan to use rodent samples, perform a pilot Western blot with mouse or rat saliva alongside human saliva to assess signal. Alternatively, sequence alignment of the immunogen region against rodent Cystatin-SA may predict epitope conservation. We have not formally validated reactivity in non-human species. For definitive rodent studies, consider species-matched antibodies or confirm binding empirically before committing to large experiments.

Can I use this Cystatin-SA antibody for immunohistochemistry on tissue sections?

Triple Point Biologics antibodies are routinely validated for immunohistochemistry, and this rabbit polyclonal is suitable for IHC on formalin-fixed paraffin-embedded or frozen sections. Start with antigen retrieval—citrate buffer pH six at sub-boiling temperature for ten minutes is a standard condition for cystatin epitopes. Use the antibody at 1:100 to 1:200 dilution and include salivary gland or esophageal tissue as a positive control, where Cystatin-SA is expressed in ductal and epithelial compartments. Optimize blocking and detection reagents for your tissue type, as endogenous biotin or high background can obscure signal in some organs.

How should I store this antibody and what is the expected shelf life?

Store the antibody at minus twenty degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The 100-microgram pack size is supplied in phosphate-buffered saline with carrier protein and preservative. Under these conditions, expect at least twelve months of stability from the manufacturing date. For long-term storage beyond one year, minus eighty degrees Celsius is preferable. Avoid storing diluted working solutions; prepare fresh dilutions in blocking buffer on the day of use. If you observe precipitate after thawing, centrifuge briefly before use rather than vortexing, which can denature immunoglobulin.

What controls should I include when working with Cystatin-SA antibody?

Include human saliva lysate as a positive control—it expresses Cystatin-SA abundantly and provides a reference band at fourteen to sixteen kilodaltons. For negative controls, use a tissue or cell line with minimal CST2 expression, such as HEK293 or lymphocytes, confirmed by quantitative PCR or prior literature. If investigating upregulation in cancer, pair tumor lysates with matched normal tissue from the same organ. For IHC, perform a no-primary-antibody control on an adjacent section to assess non-specific secondary binding. Peptide competition with the immunogen, if available, offers additional specificity confirmation but is not required for routine validation.