Anti-Cystatin-9-Like-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

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

The predicted molecular weight of CSTL1 from its 145 amino acid sequence is approximately 16 kDa. As a secreted type 2 cystatin with conserved disulfide bonds, you may see slight mobility differences between reduced and non-reduced conditions. Post-translational modifications have not been comprehensively mapped for CSTL1, but related Cres/testatin subgroup members can show glycosylation that shifts apparent MW upward by 2-5 kDa. If you observe a band substantially higher than 20 kDa, consider PNGase F treatment of your lysate to confirm whether glycoforms account for the shift.

Why don't I see CSTL1 in my HEK293 or HeLa lysates?

CSTL1 expression is restricted to reproductive tissues, primarily epididymis and testis, with minimal to absent expression in standard immortalized cell lines such as HEK293, HeLa, or U2OS. For positive controls, use human testis or epididymis lysate. If a cell line model is required, transient overexpression of CSTL1 in HEK293 followed by collection of both cell lysate and conditioned medium is appropriate, since CSTL1 carries a signal peptide and is secreted. Probe the medium fraction, as the mature secreted protein may dominate over the intracellular pool.

Does this antibody distinguish CSTL1 from other type 2 cystatins like CST8 (CRES) or CST11?

This rabbit polyclonal was raised against human CSTL1 (UniProt Q9H114). The Cres/testatin subgroup shares conserved cystatin fold residues, so cross-reactivity with CST8, CST9, CST11, or CST9L cannot be excluded by sequence alone. We have validated reactivity against human CSTL1; cross-reactivity with paralogs is predicted based on epitope homology but has not been experimentally confirmed in our hands. If your experimental question requires strict paralog discrimination, run side-by-side overexpression lysates of each paralog or include a CSTL1 knockout/knockdown control.

What dilution should I start with for Western blot?

Start at 1:1000 in your preferred blocking buffer (5% non-fat milk or 3% BSA in TBST). For low-abundance reproductive tissue samples, titrate between 1:500 and 1:2000 against a known positive lysate such as human testis. Overnight incubation at 4°C generally gives cleaner signal than 1 hour at room temperature for secreted cystatins. If background is high, increase wash stringency (0.1% to 0.2% Tween-20) before diluting the primary further. For IHC and IF, brand-wide validation supports use, but titrate independently of WB conditions.

What species does this antibody react with?

Reactivity has been validated against human CSTL1. Reactivity against mouse, rat, or other species is potential but not experimentally confirmed by us. CSTL1 orthologs in rodents fall within the rapidly evolving Cres/testatin cluster and show meaningful sequence divergence from human CSTL1, particularly in surface loops that often constitute polyclonal epitopes. If you intend to use this reagent on non-human samples, we recommend a pilot Western blot against the relevant tissue lysate (testis or epididymis) before scaling to IHC or IF applications.

What sample prep do you recommend for detecting secreted CSTL1?

Because CSTL1 is secreted, both the cellular and extracellular pools matter. For tissue, standard RIPA or NP-40 lysis of testis or epididymis works for the intracellular and membrane-associated fraction. For secreted protein in conditioned medium or seminal/epididymal fluid, concentrate 10-20x using a 3 kDa MWCO spin concentrator before loading, since baseline concentrations are typically low. Avoid serum-containing medium for secretion experiments — switch to serum-free or use a low-protein supplement for 24-48 hours before collection to prevent albumin masking the 16 kDa band.

What controls should I include for CSTL1 IHC on testis sections?

Include a no-primary control and an isotype-matched rabbit IgG control at the same protein concentration as the working antibody dilution. Peptide competition with the immunizing antigen, where feasible, is the most rigorous specificity control. For tissue context, epididymal epithelium (particularly caput and corpus regions) typically shows stronger CSTL1 signal than testicular germ cells, providing an internal positive comparison. Liver or kidney sections serve as negative tissue controls given the reproductive-restricted expression pattern. Antigen retrieval with citrate buffer (pH 6.0) is a reasonable starting point; compare against Tris-EDTA (pH 9.0) if signal is weak.

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

Upon receipt, store at -20°C for long-term use. The 100 µg format is supplied in a glycerol-containing buffer that tolerates storage at -20°C without flash-freezing. For frequent use over weeks, a working aliquot at 4°C is acceptable. Avoid repeated freeze-thaw cycles — aliquot into single-use volumes (5-10 µL) on first thaw. Polyclonal rabbit antibodies from our proteinase/inhibitor lines typically retain full activity for at least 12 months under recommended storage; signal loss in WB is the first indicator of degradation, usually preceded by increased background rather than abrupt failure.