Anti-Cystatin-A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Cystatin-A on Western blot?

Cystatin-A migrates at approximately 11 kDa on reducing SDS-PAGE, consistent with its 98-amino acid sequence. Because the protein lacks post-translational glycosylation and remains predominantly cytoplasmic, you should see a single clean band in epithelial-rich samples. In keratinocyte lysates or stratified squamous tissue homogenates, expect strong signal intensity. Degradation products occasionally appear below 11 kDa if samples were not processed quickly with protease inhibitors, given that Cystatin-A itself regulates cysteine cathepsins that can act on cellular proteins during lysis.

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

Start at 1:1000 dilution in blocking buffer for Western blot, which has been validated with this rabbit polyclonal antibody. Depending on expression levels in your sample and detection system sensitivity, the working range typically falls between 1:500 and 1:2000. Epidermal keratinocyte lysates and oral mucosa samples generally yield robust signal at 1:1000, while tissues with lower Cystatin-A expression may benefit from 1:500. Always include a positive control lysate from human epidermis or esophageal epithelium to confirm antibody performance before troubleshooting dilution with experimental samples.

Does this antibody cross-react with other cystatin family members like Cystatin-B or Cystatin-C?

This antibody was raised against Cystatin-A and shows validated specificity for human CSTA. Cystatin-B (Stefin-B) shares approximately 50 percent sequence identity with Cystatin-A within the stefin subfamily, but immunogen selection minimizes cross-reactivity risk. Cystatin-C belongs to a different subfamily, is secreted rather than cytoplasmic, and has lower homology. We have not observed cross-reactivity with Cystatins B or C in validation experiments, but if you work with samples expressing multiple stefins at high levels, consider running recombinant Cystatin-B alongside your samples as a specificity control.

Which human tissues or cell lines serve as good positive controls for Cystatin-A expression?

Primary human keratinocytes, HaCaT cells, or tissue lysates from stratified squamous epithelia provide reliable positive controls. Cystatin-A is highly abundant in epidermis, particularly in the lower spinous and basal layers, as well as in esophageal and oral mucosal epithelium. If working with cell lines, HaCaT spontaneously immortalized keratinocytes express robust Cystatin-A. Avoid using fibroblasts, lymphocytes, or most mesenchymal cell types as positive controls since expression is minimal. For IHC, normal human skin sections serve as an ideal positive tissue control, with expected cytoplasmic staining in keratinocytes.

Is this Cystatin-A antibody validated for mouse or rat samples?

This antibody has been validated for human samples. Cross-reactivity with mouse or rat Cystatin-A is predicted based on sequence homology—murine and rat orthologs share approximately 70 to 75 percent identity with human CSTA—but we have not formally validated reactivity in these species. If you plan to use mouse or rat samples, treat this as experimental and include appropriate positive controls such as mouse epidermis or esophageal tissue. Test at the recommended 1:1000 dilution initially, and verify band size and specificity with a blocking peptide or siRNA knockdown if signal is ambiguous.

Can I use this antibody for immunofluorescence or immunohistochemistry on paraffin sections?

Yes, Triple Point Biologics antibodies are validated for Western blot; immunofluorescence and IHC application data is in progress in addition to Western blot. For IHC on formalin-fixed paraffin-embedded sections, antigen retrieval is typically required—citrate buffer at pH 6.0 with heat-mediated retrieval works well for Cystatin-A. Expect cytoplasmic staining in keratinocytes and stratified squamous epithelia. Start with a 1:100 to 1:200 dilution for IHC and IF; optimal concentration depends on your detection system and tissue fixation time. Include adjacent serial sections as negative controls using preimmune rabbit IgG at matched concentration.

Should I add protease inhibitors when preparing lysates for Cystatin-A Western blot?

Yes, include a broad-spectrum protease inhibitor cocktail during lysis. Although Cystatin-A is a protease inhibitor itself, it specifically targets cysteine cathepsins, and your sample will contain serine, aspartyl, and metalloproteases that can degrade cellular proteins during extraction. Cysteine cathepsins released during cell disruption may also act on other targets before Cystatin-A re-equilibrates in dilute lysate. Work quickly on ice, snap-freeze samples if not analyzing immediately, and avoid multiple freeze-thaw cycles. This practice ensures you detect intact 11 kDa Cystatin-A rather than degradation fragments.

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

Store the antibody at -20°C in undiluted aliquots to maintain long-term stability. Avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregate formation. For frequent use, prepare working aliquots and keep one at 4°C with 0.02 to 0.05 percent sodium azide as preservative for up to one month. The 100 µg pack size is suitable for extensive experimental series. Polyclonal antibodies from immunized rabbits, when stored properly at -20°C, typically retain activity for two to three years from production date. Check the product vial label for the specific lot expiration date.