Anti-Cystatin-B Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Cystatin-B on Western blot?

Cystatin-B migrates at approximately 11 kDa on Western blot, consistent with its 98-amino acid sequence. The protein lacks post-translational cleavage and runs close to its predicted mass. Some researchers report a band slightly higher at 13-14 kDa depending on gel percentage and running conditions. Because CSTB is a small, basic protein, we recommend using 15% or higher acrylamide gels for optimal resolution. Avoid over-reducing samples, as Cystatin-B contains cysteine residues important for its native conformation, though standard reducing conditions are generally compatible.

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

We validate this antibody at 1:1000 for Western blot. This is a suitable starting dilution for most mammalian cell lysates and tissue extracts where Cystatin-B is reasonably abundant. For samples with lower expression—such as certain primary cell types—you may need to titre down to 1:500. Conversely, in liver or immune cell lysates where CSTB is highly expressed, 1:2000 often provides clean signal with reduced background. Always include a titration series with your specific sample type and detection system to determine optimal working dilution.

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

This antibody is raised against Cystatin-B and has been validated for human CSTB. The cystatin superfamily shares structural homology, particularly within the stefin subfamily, so cross-reactivity with Cystatin-A (stefin-A) is theoretically possible though not systematically tested in our hands. If you are working in samples expressing multiple stefins or secreted cystatins, we recommend running recombinant Cystatin-A and Cystatin-C alongside your samples as specificity controls. Knock-down or knockout lysates, where available, remain the most definitive specificity validation.

Will this antibody work in mouse or rat samples?

The antibody is validated against human Cystatin-B. Mouse and rat CSTB share approximately 85-90% sequence identity with human, which often permits cross-reactivity for polyclonal antibodies, but we have not formally validated reactivity in these species. If you plan to use rodent samples, treat cross-reactivity as predicted rather than confirmed. We recommend running a positive control human lysate in parallel and verifying that your rodent band migrates at the expected size. Researchers in our field have reported variable success with cross-species use of CSTB antibodies.

Can I use this antibody for immunohistochemistry on paraffin sections?

Triple Point Biologics antibodies are routinely validated for IHC applications, and this format is compatible with paraffin-embedded tissue. For Cystatin-B, antigen retrieval is typically required—citrate buffer pH 6.0 with heat-induced epitope retrieval is a standard starting point. Because CSTB is an intracellular protein, you should expect diffuse cytoplasmic staining. Liver, spleen, and brain sections serve as useful positive-tissue controls given the known distribution of Cystatin-B. As with any IHC experiment, include appropriate isotype and no-primary controls to assess background.

What are good positive and negative control samples for Cystatin-B expression?

Cystatin-B is ubiquitously expressed but particularly abundant in liver, so human liver lysate or hepatocyte-derived cell lines such as HepG2 serve as strong positive controls. Peripheral blood mononuclear cells and brain tissue also express high levels. For a functional validation, consider comparing lysates from wild-type cells to CSTB knockout or knockdown samples if available. There are no truly CSTB-negative human tissues under normal conditions, but expression levels vary. Low-expression samples include certain differentiated epithelial lines; consult expression databases such as Human Protein Atlas for tissue-specific guidance.

How should I store the antibody and how long is it stable?

Store the antibody at -20°C as supplied. For long-term storage beyond six months, aliquot upon receipt to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month with 0.02% sodium azide as preservative. Avoid storing diluted antibody; prepare fresh working dilutions in blocking buffer on the day of use. Properly stored aliquots typically retain activity for two years or more, though we recommend titration if the antibody has been stored beyond the expiration date.

Why do I see multiple bands on Western blot with this Cystatin-B antibody?

Cystatin-B should appear as a single band near 11 kDa. Multiple bands may indicate degradation, particularly if you are working with aged samples or tissue lysates rich in cathepsins—the very proteases CSTB inhibits. Ensure you add protease inhibitors immediately during lysis and keep samples cold. A higher molecular weight band around 22 kDa occasionally appears and may represent a dimer, as CSTB can form non-covalent oligomers under certain conditions. Non-specific bands above 20 kDa are more likely off-target binding; try increasing your blocking time or raising your primary antibody dilution to 1:2000.