Anti-Cystatin-C Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Mature Cystatin-C typically migrates at approximately 13-14 kDa on reducing SDS-PAGE, corresponding to the secreted form after signal peptide cleavage. The full-length precursor with the 26-residue signal peptide is rarely observed in extracellular samples. If you are working with conditioned media or serum, expect a single band around 13 kDa. In lysates from cells overexpressing CST3, you may occasionally detect the pre-protein at approximately 16 kDa, though the mature form predominates. Glycosylation does not significantly alter migration for this protein.

What starting dilution should I use for Western blot with this Cystatin-C antibody?

We recommend starting at 1:1000 for Western blot applications, which has been validated with this antibody. Depending on your sample type and Cystatin-C expression level, the working dilution may range from 1:500 to 1:2000. Serum and conditioned media typically yield strong signals at 1:1000 to 1:2000 due to high constitutive secretion. For whole cell lysates, start at 1:1000 and optimize based on signal intensity and background. Allow overnight incubation at 4°C for best results with polyclonal antibodies targeting this abundant secreted protein.

Does this antibody cross-react with mouse or rat Cystatin-C?

This antibody has been validated for human Cystatin-C. Cross-reactivity with mouse or rat Cystatin-C is predicted based on sequence homology but has not been experimentally validated by Triple Point Biologics. Human CST3 shares approximately 75-80 percent sequence identity with mouse and rat orthologs, suggesting potential cross-reactivity. If you are working with rodent samples, we recommend testing the antibody at the suggested starting dilution and including a positive control with recombinant human Cystatin-C to confirm specificity. Validation in your specific model system is advisable before committing to a full experiment series.

What positive and negative controls should I use when detecting Cystatin-C?

For positive controls, HEK293 or HeLa conditioned media are reliable sources, as Cystatin-C is constitutively secreted by most nucleated cells. Recombinant human Cystatin-C provides a defined molecular weight reference. Human serum or plasma also contains Cystatin-C at approximately 0.5-1.0 mg/L. For negative controls, bacterial lysates are suitable, as prokaryotes do not express cystatin family members. Alternatively, use lysates from cells after CST3 knockdown if your experimental design permits. Red blood cells, which lack nuclei, express minimal Cystatin-C and can serve as a low-expression control in certain contexts.

Can I use this antibody for immunohistochemistry on tissue sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry applications in addition to Western blot. For IHC with Cystatin-C, antigen retrieval is typically required due to formalin-induced crosslinking. Citrate buffer (pH 6.0) heat-induced epitope retrieval is a standard starting point. Cystatin-C is widely expressed, so expect signal in most nucleated cell types, with particularly strong staining in kidney tubular epithelium, neurons, and various secretory cells. Starting dilutions for IHC are generally higher concentration than Western blot—begin optimization at 1:100 to 1:200 and titre accordingly for your tissue type and fixation protocol.

Will this antibody detect the pro-form of Cystatin-C with the signal peptide attached?

The epitope recognized by this polyclonal antibody has not been mapped to a specific region, so reactivity with the uncleaved pre-protein is theoretically possible but unlikely to be prominent. The signal peptide is rapidly cleaved in the endoplasmic reticulum during normal biosynthesis, and the mature secreted form predominates in virtually all biological samples. If you are specifically investigating signal peptide processing or using proteasome inhibitors that might stabilize precursor forms, expect the mature 13 kDa band to remain the dominant species. For studies focused exclusively on the pro-form, a peptide-specific antibody targeting the signal sequence would be more appropriate.

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

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise polyclonal antibody performance. The 100 µg pack size is suitable for aliquoting into 10-20 µL volumes depending on your typical experiment frequency. Once thawed, aliquots can be kept at 4°C for up to one month for convenience during active experiments. Avoid prolonged storage above 4°C. Under proper storage conditions at -20°C, rabbit polyclonal antibodies typically maintain activity for at least two years from manufacture. Addition of glycerol or BSA as stabilizers is generally unnecessary for this product as supplied.

Why am I seeing multiple bands on my Cystatin-C Western blot?

A single band at 13-14 kDa is expected for mature Cystatin-C. Multiple bands may indicate degradation, particularly if you are analyzing aged serum samples or lysates without protease inhibitors—ironic, given Cystatin-C's role as a protease inhibitor. Ensure samples are prepared with fresh protease inhibitor cocktails and processed quickly. Higher molecular weight bands may represent complexes with target proteases, though these typically dissociate under reducing conditions. Lower molecular weight fragments suggest proteolytic clipping. If working with clinical samples, pre-analytical variables including collection and storage conditions significantly affect Cystatin-C integrity. Include freshly prepared positive controls to distinguish sample-related issues from antibody performance.