Anti-Cystatin-F Rabbit Polyclonal Antibody

Frequently Asked Questions

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

The mature secreted form of Cystatin-F runs at approximately 16 kDa on reducing SDS-PAGE, corresponding to the 145-amino-acid processed protein. You may also detect a band near 18 kDa representing the glycosylated precursor form, depending on your cell type and lysis conditions. Cystatin-F undergoes signal peptide cleavage and contains a disulfide bond, so reduction is recommended for optimal detection. If working with conditioned media or serum, expect predominantly the mature 16 kDa form. Intracellular lysates from hematopoietic cells may show both precursor and mature species.

Why is Cystatin-F expression so low in my lysates compared to Cystatin C?

Cystatin-F expression is restricted to hematopoietic and immune cells, unlike the constitutively expressed Cystatin C found in nearly all tissues. Even within immune compartments, basal expression can be modest. If you are working with non-hematopoietic cell lines or primary tissues lacking lymphoid populations, Cystatin-F may be undetectable. Consider using activated T cells, NK cells, or dendritic cells as positive controls. Additionally, because Cystatin-F is secreted, a significant fraction partitions into culture supernatant rather than remaining cell-associated, which can further reduce signal in whole-cell lysates.

What starting dilution should I use for Cystatin-F Western blots?

We recommend starting at 1:1000 for Western blot with standard chemiluminescent detection, based on validation with human hematopoietic cell lysates. If signal is weak or your sample has low Cystatin-F expression, titrate upward to 1:500 or 1:250. Conversely, if working with concentrated conditioned media or purified protein, you may achieve clean results at 1:2000. Always include a no-primary control to assess nonspecific binding. Overnight incubation at 4°C often improves sensitivity compared to one-hour room-temperature protocols for this target.

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

This antibody has been validated against human Cystatin-F. Cross-reactivity with mouse or rat orthologs is predicted based on sequence homology in the immunogen region but has not been experimentally confirmed. Human and mouse CST7 share approximately 70% amino acid identity, which suggests possible cross-reactivity, though affinity may be reduced. If you plan to use mouse or rat samples, we recommend running a pilot blot with known positive control lysate from the target species and verifying band position at the expected molecular weight before proceeding with experimental samples.

What are appropriate positive and negative controls for Cystatin-F experiments?

For positive controls, use lysates from activated human T cells, NK cell lines such as NK-92, or monocyte-derived dendritic cells, all of which express CST7 at detectable levels. Conditioned media from these cells can provide stronger signal due to secretion. Appropriate negative controls include non-hematopoietic cell lines such as HeLa, HEK293, or primary fibroblasts, which typically lack Cystatin-F expression. For IHC, human spleen or lymph node sections serve as excellent positive tissue controls. Including a no-primary antibody control in parallel lanes or sections helps confirm specificity.

Can I use this Cystatin-F antibody for immunofluorescence or IHC?

This antibody has been validated for Western blot; IHC validation is in progress and can be used for immunofluorescence applications. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required; citrate buffer pH 6.0 with heat-induced epitope retrieval is a reasonable starting point. For immunofluorescence on fixed cells, standard methanol or paraformaldehyde fixation should preserve the epitope. Because Cystatin-F is secreted, expect both intracellular staining in the ER-Golgi compartment and extracellular or membrane-proximal signal depending on cell type and stimulation state. Titrate antibody concentration starting around 1-2 µg/ml.

How should I store the Cystatin-F antibody and what is its shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied at 100 µg and remains stable for at least 12 months under these conditions. For short-term use within two to three weeks, storing at 4°C is acceptable. Do not store diluted antibody; prepare working solutions fresh for each experiment. If you observe increased background or reduced signal over time, aggregation may have occurred. Centrifuge thawed aliquots briefly before use to pellet any precipitate.

Does Cystatin-F exist as multiple isoforms or splice variants?

CST7 does not have well-characterized splice variants that produce functionally distinct protein isoforms. The primary transcript encodes a single precursor protein that undergoes signal peptide cleavage and disulfide bond formation to yield the mature 145-amino-acid secreted form. Post-translational modifications such as glycosylation can result in minor mobility shifts on SDS-PAGE. Unlike some cystatin family members, alternative splicing does not appear to generate additional complexity for this target. Therefore, you should expect a single dominant band corresponding to the mature protein under reducing conditions.