Anti-Cystatin-9 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Cystatin-9 on Western blot?

Cystatin-9 runs at approximately 17-18 kDa under reducing conditions, corresponding to the mature secreted protein of 159 amino acids after signal peptide cleavage. The predicted molecular weight from sequence is ~17 kDa. Because CST9 is a secreted cystatin, you may observe slightly diffuse migration depending on sample type and any post-translational modifications. In cell lysates expressing low endogenous levels, concentration or enrichment of conditioned media may be necessary. Standard sample preparation with reducing agent and boiling is appropriate for this target.

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

Cross-reactivity with other type 2 cystatins has not been systematically characterized for this polyclonal reagent. The cystatin superfamily shares significant structural homology, particularly within the conserved cystatin fold. We recommend including samples known to express related cystatins (such as cystatin C, D, or SN) as specificity controls if your experimental system co-expresses multiple family members. Pre-absorption or siRNA knockdown controls are advisable when distinguishing CST9 signal from related proteins in complex tissues, particularly in testis or immune cell lysates where multiple cystatins are co-expressed.

What dilution should I start with for Western blot of Cystatin-9?

We validate this antibody at 1:1000 for Western blot. This serves as a reliable starting point for most applications with standard protein loads of 20-50 µg per lane. Because endogenous CST9 expression is relatively restricted and can be low in many cell types, you may need to optimize upward to 1:500 if signal is weak, or extend exposure time. For recombinant CST9 or overexpression systems, 1:2000 to 1:5000 may be sufficient. Titre the antibody against your specific sample type and detection system for optimal signal-to-noise.

Which tissues or cell lines are good positive controls for Cystatin-9 expression?

Testis tissue is the most reliable positive control for CST9, where expression is well-documented. Among cell lines, transcript data suggest hematopoietic and immune-related lines may express CST9, though protein-level validation is limited in the literature. If working with cell lines, consider screening several candidates by RT-PCR before committing to Western blot optimization. Recombinant CST9 protein is advisable as a reference standard to confirm antibody recognition and establish expected migration. Negative controls from tissues with undetectable CST9 transcript, such as liver or skeletal muscle, help assess specificity.

Is this Cystatin-9 antibody validated in species other than human?

This antibody is validated specifically for human Cystatin-9. Predicted cross-reactivity with other species depends on sequence homology in the epitope region, which has not been empirically tested. If you are working with mouse, rat, or other model organisms, alignment of the CST9 ortholog sequence against human is advisable before proceeding. We cannot guarantee performance in non-human samples without direct validation. For non-human studies, consider titrating the antibody across a dilution series and including both positive control lysates and blocking peptide controls to assess specificity empirically.

Can I use this antibody for immunohistochemistry or immunofluorescence?

Triple Point Biologics antibodies are routinely validated for IHC and immunofluorescence in addition to Western blot. While we have not published specific protocols for CST9 IHC, the reagent is suitable for testing in fixed tissue sections. Start with standard antigen retrieval protocols (citrate buffer, pH 6.0, or Tris-EDTA, pH 9.0) and an antibody dilution of 1:100 to 1:200. Because CST9 is a secreted protein, expect diffuse extracellular or perinuclear Golgi-associated staining in expressing cells. Validate staining patterns with known CST9-positive tissue such as testis and include isotype or pre-immune controls.

What is the expected subcellular localization of Cystatin-9?

Cystatin-9 is a secreted protein with an N-terminal signal peptide that directs it through the classical secretory pathway. In producing cells, you may observe CST9 signal in the endoplasmic reticulum and Golgi before secretion. Once secreted, CST9 localizes extracellularly, where it presumably functions as a cysteine protease inhibitor. In Western blots of cell lysates, you will detect intracellular precursor and mature forms; conditioned media from cultured cells will contain the secreted mature protein. For immunofluorescence, expect perinuclear and secretory vesicle staining in expressing cells, with extracellular accumulation depending on culture conditions.

How should I store this Cystatin-9 antibody and what is the shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise titre and increase background. The antibody is supplied in a stabilized formulation and typically remains stable for at least 12 months from the date of receipt when stored properly. Once thawed, an aliquot can be held at 4°C for up to one month for convenient repeated use. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer immediately before use. If you observe increased background or loss of signal over time, prepare a fresh aliquot from frozen stock.