Anti-Cystatin-D Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cystatin-D migrates at approximately 14-16 kDa on reducing SDS-PAGE, corresponding to its 142-amino acid mature secreted form. The predicted molecular weight from sequence is 14.3 kDa. Because Cystatin-D is a secreted protein that undergoes signal peptide cleavage, you will not observe the full-length precursor under most conditions. If working with saliva or conditioned media, expect a single prominent band in this range. Post-translational modifications are minimal for this cystatin family member, so band shifts are uncommon unless samples have undergone significant degradation.

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

This antibody was raised against human Cystatin-D and has been validated specifically for CST5 gene product. Cross-reactivity with other cystatins (A, B, C, S, SA, SN) has not been systematically tested. Given the sequence divergence among cystatin family members, particularly in surface-exposed epitopes, cross-reactivity is unlikely but cannot be ruled out without direct testing. If you are working in tissues or fluids that co-express multiple cystatins, such as saliva containing Cystatins S, SA, and SN, consider including lysates from cells transfected with individual cystatin isoforms as specificity controls.

What is the recommended starting dilution for Western blot with this Cystatin-D antibody?

The recommended starting dilution is 1:1000 for Western blot applications. This dilution has been validated with human samples. Because Cystatin-D is highly abundant in saliva but expressed at lower levels in most tissues, optimal dilution may vary depending on sample source. For saliva or oral fluid samples, you may achieve good signal at 1:2000 or even 1:5000. For tissue lysates or serum, start at 1:1000 and titrate upward to 1:500 if signal is weak. Always include a positive control such as human saliva or parotid gland lysate.

Will this antibody detect Cystatin-D in mouse or rat samples?

This antibody has been validated only for human Cystatin-D. Cross-reactivity with mouse or rat Cystatin-D is predicted based on sequence homology but has not been experimentally confirmed. Human CST5 shares approximately 70-75 percent identity with rodent orthologs, which may be sufficient for cross-reaction depending on the epitope recognized by this polyclonal. If working with rodent samples, treat results as preliminary until confirmed by alternative methods such as mass spectrometry or a species-specific antibody. Consider running human saliva as a positive control alongside rodent samples to assess signal specificity.

What positive control samples should I use for Cystatin-D detection?

Human saliva is the most reliable positive control, as Cystatin-D is one of the most abundant salivary proteins. Whole saliva can be diluted 1:100 to 1:1000 in sample buffer for Western blot. Parotid or submandibular salivary gland tissue lysates also serve as strong positive controls. Serum contains Cystatin-D at much lower levels and is not recommended as a primary control. For IHC, normal human salivary gland tissue sections provide excellent signal. Avoid using non-oral tissues as positive controls unless you have independent evidence of Cystatin-D expression in your specific experimental context.

Can I use this antibody for immunofluorescence or immunohistochemistry?

Yes, Triple Point Biologics antibodies are validated across Western blot platforms. 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 standard starting condition. For immunofluorescence, the antibody can be used on fixed cells or frozen tissue sections. Start with a 1:100 to 1:200 dilution and optimize based on signal-to-background ratio. Because Cystatin-D is a secreted protein, expect diffuse extracellular or vesicular staining patterns rather than distinct subcellular localization in producing cells.

How should I store the Cystatin-D antibody and what is its stability?

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 as a rabbit polyclonal in solution and is stable for at least 12 months from date of receipt when stored properly. For frequent use, keep a working aliquot at 4°C for up to one month; do not store diluted antibody for more than one week. Avoid prolonged exposure to room temperature. Do not add sodium azide if you plan to use the antibody for applications involving enzymatic detection systems, as azide inhibits horseradish peroxidase.

Why am I not detecting Cystatin-D in serum or plasma by Western blot?

Cystatin-D is present in serum and plasma at much lower concentrations than in saliva, often below the detection limit of standard Western blot. While Cystatin-D is secreted and does enter circulation, it is not a high-abundance serum protein like Cystatin-C. If detection in serum is essential, consider concentrating your sample by acetone or TCA precipitation, using at least 50-100 micrograms total protein per lane, or switching to a more sensitive detection method such as ELISA. Confirm that your positive control (saliva) produces the expected band at approximately 14-16 kDa before troubleshooting serum samples.