Anti-Testisin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Testisin (PRSS21) has a predicted molecular weight of approximately 35 kDa based on its 314 amino acid sequence. However, the mature protein after signal peptide cleavage is typically observed around 32-33 kDa on reducing SDS-PAGE. Because Testisin contains a propeptide domain that may be cleaved during activation, you may see multiple bands representing zymogen and processed forms depending on your tissue or cell lysate. Testicular germ cell lysates often show the predominant band near 33 kDa. Always include a positive control lysate from testis or a Testisin-transfected cell line to confirm the expected migration pattern.

What is the recommended starting dilution for Testisin antibody in Western blot?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal Testisin antibody. This dilution has been validated with human testis lysate and should provide robust signal for samples expressing physiological levels of Testisin. If you are working with overexpression systems or concentrated samples, you may titrate up to 1:2000. Conversely, samples with low Testisin expression, such as certain eosinophil preparations, may require optimization down to 1:500. Incubate overnight at 4°C in 5% non-fat milk or BSA in TBST for optimal signal-to-noise ratio.

Does this Testisin antibody cross-react with other trypsin-like serine proteases?

This polyclonal antibody was raised against a Testisin-specific immunogen and has been validated for human Testisin (PRSS21). Given that the trypsin-like serine protease family shares conserved catalytic domains, some cross-reactivity with closely related proteases is theoretically possible but has not been systematically tested. We have not observed cross-reactivity with common serine proteases like trypsin, thrombin, or kallikreins in validation experiments using testis lysate. If you are working with complex tissue extracts containing multiple serine proteases, include appropriate negative controls such as PRSS21 knockout lysates or siRNA-depleted samples to confirm band specificity.

What tissues or cell types express Testisin at detectable levels?

Testisin is predominantly expressed in testicular germ cells, particularly during spermatogenesis, making testis tissue the primary positive control for antibody validation. Expression is highest in spermatocytes and round spermatids. Outside the testis, Testisin has been identified in eosinophils where it functions as eosinophil serine protease 1 (ESP-1), though expression levels are typically lower than in testis. Most other adult tissues show minimal to undetectable Testisin expression by Western blot. If investigating testicular germ cell tumors, seminomas and some non-seminomatous tumors may retain expression depending on differentiation state. Standard cell lines rarely express endogenous Testisin unless of germ cell origin.

Can I use this antibody for immunohistochemistry or immunofluorescence on testis sections?

Yes, Triple Point Biologics antibodies are validated for Western blot; immunohistochemistry and immunofluorescence validation is in progress. For IHC on paraffin-embedded testis sections, perform antigen retrieval using citrate buffer pH 6.0 with heat-induced epitope retrieval. Start with a 1:100 to 1:200 dilution and optimize based on signal intensity. Testisin staining should localize to the membrane of germ cells, particularly spermatocytes and round spermatids within seminiferous tubules. For immunofluorescence on frozen sections or cultured germ cells, fix with 4% paraformaldehyde and permeabilize appropriately. Expect membrane and possible cytoplasmic staining patterns depending on processing state.

Will this human-validated Testisin antibody work with mouse or rat samples?

This antibody has been validated specifically for human Testisin and reactivity with mouse or rat orthologs is predicted but not experimentally confirmed. Human PRSS21 shares approximately 70-75% sequence identity with mouse and rat Testisin, with conservation highest in the catalytic domain. Cross-reactivity is plausible, particularly for Western blot applications, but we recommend empirical testing with appropriate positive controls such as mouse or rat testis lysate. Start at the recommended 1:1000 dilution and include human testis lysate as a reference standard. If you require guaranteed rodent reactivity, contact us to discuss species-specific antibody options or validation data that may be available.

How should I prepare testis lysate to preserve Testisin for Western blot analysis?

Testisin is a serine protease and can undergo autocatalytic processing or degradation if protease inhibitors are not included during lysis. Homogenize testis tissue immediately in cold RIPA or NP-40 lysis buffer supplemented with a broad-spectrum protease inhibitor cocktail containing serine protease inhibitors such as AEBSF or PMSF. Keep samples on ice throughout and avoid repeated freeze-thaw cycles, which can promote degradation. Process tissue quickly and snap-freeze aliquots in liquid nitrogen if not using immediately. Some researchers add 1 mM benzamidine for additional serine protease inhibition. Proper inhibitor use will preserve both zymogen and processed forms of Testisin for accurate Western blot characterization.

What is the proper storage condition for this Testisin antibody and how long is it stable?

Store the antibody at -20°C in the glycerol-containing storage buffer provided. Under these conditions, the antibody remains stable for at least 12 months from the date of receipt. For routine use, aliquot the antibody into single-use volumes to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation over time. If you use the antibody frequently, a working aliquot can be stored at 4°C for up to one month. Do not store diluted antibody in blocking buffer for extended periods. Centrifuge thawed aliquots briefly before use to pellet any aggregates that may have formed during storage.