Anti-Cystatin-Like 1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for CSTL1 on a Western blot?

CSTL1 is a 145-amino acid secreted protein with a predicted molecular weight of approximately 16 kDa. Because it is secreted and may undergo post-translational modifications including glycosylation, you may observe the band slightly higher than predicted, typically in the 16-20 kDa range. If working with conditioned media or serum samples, confirm your sample preparation retains secreted proteins. For initial experiments, run a molecular weight marker covering 10-25 kDa and consider both reducing and non-reducing conditions to assess any disulfide-linked dimers, though CSTL1 is generally monomeric.

What dilution should I start with for Western blot with this CSTL1 antibody?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this antibody. Depending on CSTL1 expression levels in your sample type, you may need to optimize between 1:500 and 1:2000. For reproductive tissues or testis lysates where CSTL1 is more abundant, 1:1000 or higher dilutions typically work well. For cell lines or tissues with lower expression, begin at 1:500. Incubate overnight at 4°C in 5% non-fat milk or BSA in TBST. Always include a positive control lysate from testis or known CSTL1-expressing cells.

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

CSTL1 shares structural homology with other type 2 cystatins, particularly the reproductive cystatin subgroup including Cres/Testatin and Cystatin-C. While this rabbit polyclonal was raised against CSTL1-specific epitopes, cross-reactivity with closely related family members has not been exhaustively tested. If you are working with samples expressing multiple cystatins, consider running a parallel blot with recombinant Cystatin-C or other family members to assess specificity. The restricted tissue expression of CSTL1 compared to ubiquitous cystatins like Cystatin-C can help with interpretation in reproductive tissue lysates.

Will this antibody work for mouse or rat samples?

This antibody is validated for human CSTL1. Cross-reactivity with mouse or rat orthologs is predicted but not experimentally validated. Human CSTL1 shows moderate sequence conservation with rodent orthologs, particularly in the cystatin domain, so cross-reactivity is plausible. If you are working with mouse or rat samples, we recommend testing the antibody at the suggested 1:1000 dilution first, using testis lysate as a positive control. Include human samples as a reference if possible. Be prepared to optimize dilution or blocking conditions, and validate any observed bands by peptide competition or siRNA knockdown.

What are good positive and negative control tissues for CSTL1 expression?

CSTL1 is part of the reproductive cystatin subgroup with tissue-restricted expression. Human testis is the most reliable positive control, as CSTL1 shows prominent expression in reproductive tissues. Epididymis and potentially prostate may also express CSTL1, though levels vary. For negative controls, use tissues with minimal reproductive cystatin expression such as liver, brain, or skeletal muscle. If working with cell lines, consider testis-derived or germ cell lines as potential positives. Always confirm your positive control shows the expected 16-20 kDa band before troubleshooting experimental samples.

Can I use this antibody for immunohistochemistry on paraffin sections?

Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress. For paraffin-embedded sections, standard antigen retrieval is typically required. Begin with citrate buffer (pH 6.0) heat-mediated retrieval and test the antibody at dilutions between 1:100 and 1:500. CSTL1 is a secreted protein, so you may observe both intracellular staining in expressing cells and extracellular signal in interstitial spaces, particularly in testis sections. Include appropriate secondary-only controls and consider testing on frozen sections first if you encounter high background, as secreted proteins can sometimes show artifactual localization after fixation.

How should I store this antibody and what is the expected stability?

Store the antibody at -20°C as supplied. For frequent use, aliquot into single-use volumes to avoid repeated freeze-thaw cycles, which can reduce antibody activity over time. Once aliquoted, the antibody remains stable at -20°C for at least 12 months. For working aliquots, short-term storage at 4°C for up to one month is acceptable if sodium azide (0.02-0.05%) is added as a preservative. Avoid prolonged exposure to room temperature and never store diluted antibody in working buffer for more than a week. If you observe declining signal over time, prepare a fresh aliquot.

Why am I not detecting CSTL1 in my cell culture media by Western blot?

CSTL1 is a secreted protein, but detection in conditioned media depends on several factors. First, confirm your cell line expresses CSTL1 by checking mRNA levels or analyzing cell lysates directly. Many common cell lines have minimal CSTL1 expression outside reproductive contexts. Second, secreted protein concentration in media is often low; consider concentrating conditioned media 10-20-fold using centrifugal filters (3-10 kDa cutoff). Third, serum proteins in culture media can interfere—try serum-free conditions for 24-48 hours before harvesting. Finally, verify that CSTL1 is stable in your media conditions; some secreted proteins require protease inhibitors during collection.