Anti-Cystatin-M Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected band size for Cystatin-M on Western blot?

Cystatin-M (CST6) migrates at approximately 14-16 kDa on reducing SDS-PAGE, corresponding to the 149-amino-acid mature protein. The calculated molecular weight is 16.5 kDa, though migration may vary slightly depending on post-translational modifications and gel conditions. Because Cystatin-M is a secreted protein, you may observe the signal-peptide-containing precursor form at a slightly higher molecular weight in lysates from cells that actively synthesize it. Conditioned media from expressing cells typically shows the mature, processed form. If you see a doublet, consider that glycosylation or incomplete signal peptide cleavage may contribute.

What sample types work best for detecting Cystatin-M with this antibody?

This antibody performs reliably in lysates from stratified squamous epithelia, cultured keratinocytes, and relevant tumor cell lines that retain CST6 expression. Because Cystatin-M is secreted, conditioned media from expressing cells often yields cleaner bands at lower backgrounds than whole-cell lysates. For IHC, formalin-fixed paraffin-embedded sections of skin, hair follicles, sweat glands, and mammary epithelium provide physiologically relevant positive controls. Note that CST6 is frequently downregulated or silenced in several carcinomas, so verify expression in your model system by qPCR or published datasets before expecting robust signal.

Should I start at the recommended 1:1000 dilution for Western blot?

The recommended 1:1000 dilution is a validated starting point for lysates with moderate to high Cystatin-M expression. If you are working with conditioned media, skin tissue lysates, or keratinocyte lines known to express CST6, begin at 1:1000. For samples with uncertain or low expression, such as cancer cell lines where CST6 may be epigenetically silenced, consider starting at 1:500 and titrating upward to 1:2000 after assessing signal strength. Because this is a rabbit polyclonal, batch-to-batch titer variations are minimal but not absent; pilot a dilution series with your specific lysate.

Does this antibody cross-react with other cystatins on Western blot?

This polyclonal was raised against Cystatin-M and validated for specificity to CST6. The type 2 cystatin family shares structural homology, so cross-reactivity with closely related members such as Cystatin-A, -B, -C, or -D is theoretically possible but has not been systematically reported with this reagent. To assess specificity in your system, run parallel blots with recombinant Cystatin-M alongside lysates from tissues expressing other cystatins. If you see unexpected bands, RNAi-mediated knockdown of CST6 or comparison with a second CST6 antibody targeting a non-overlapping epitope will help confirm identity.

Is this antibody predicted to work with mouse or rat samples?

This antibody is validated for human Cystatin-M. Cross-reactivity with mouse or rat orthologs is not validated and should be considered uncertain. Human CST6 shares approximately 60-70 percent amino acid identity with rodent orthologs, so epitope conservation is not guaranteed. If you need to detect Cystatin-M in rodent samples, test the antibody empirically at the recommended dilution alongside a known human positive control. Alternatively, sourcing a reagent specifically raised against the rodent sequence will reduce experimental ambiguity. We have not performed formal cross-species validation for this polyclonal.

What positive and negative control samples should I use for Cystatin-M?

For positive controls, use lysates or conditioned media from primary human keratinocytes, HaCaT cells, or skin tissue, all of which express CST6 endogenously. Normal human epidermis sections serve as excellent positive controls for IHC. For negative controls, select cell lines or tissues where CST6 is known to be absent or silenced, such as many breast or prostate carcinoma lines. Recombinant human Cystatin-M protein, if available, provides an unambiguous reference for molecular weight and antibody specificity. Blocking-peptide competition or CST6-knockout lysates offer additional validation when needed.

How should I store this Cystatin-M antibody to maintain activity?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The supplied format is typically in a glycerol-containing buffer that remains liquid at -20°C, allowing direct pipetting if you prefer not to aliquot. For short-term use over a few weeks, storing at 4°C is acceptable. Avoid prolonged exposure to room temperature. Do not freeze-thaw more than three times. If you observe particulate matter after thawing, centrifuge briefly before use to pellet aggregates that may increase background in immunofluorescence or IHC.

Why might I see weak or absent signal for Cystatin-M in cancer cell lines?

CST6 is frequently downregulated or epigenetically silenced in several epithelial cancers, including breast, lung, and prostate carcinomas, often via promoter hypermethylation. Loss of Cystatin-M expression correlates with increased invasiveness in multiple tumor models. Before troubleshooting antibody performance, confirm that your cell line or tumor sample actually expresses CST6 at the mRNA level using qPCR or RNA-seq databases. If expression is absent or very low, no antibody dilution adjustment will yield signal. Conversely, treatment with demethylating agents can restore CST6 expression in some silenced lines, providing an inducible positive control.