Anti-HTRA-4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for HTRA-4 on a Western blot?

Full-length HTRA-4 runs at approximately 52 kDa on SDS-PAGE, corresponding to its 476-amino acid sequence. However, because HTRA-4 is a secreted protease with signal peptide cleavage and potential glycosylation in placental tissues, you may observe a band closer to 55-60 kDa depending on post-translational modifications. If working with conditioned media or extracellular matrix fractions, processed forms or proteolytic fragments may also appear at lower molecular weights. Always include a positive control lysate from placental tissue or transfected cells expressing HTRA-4 to confirm band identity.

What starting dilution do you recommend for HTRA-4 Western blot?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this antibody. Depending on HTRA-4 expression levels in your sample, you may need to optimize between 1:500 and 1:2000. Placental lysates typically yield strong signal at 1:1000, while tissues with lower endogenous expression may require more concentrated antibody. Use standard blocking conditions with five percent non-fat milk or BSA in TBST, and incubate overnight at four degrees Celsius for optimal results. Always titrate against your specific sample type to minimize background.

Is this HTRA-4 antibody suitable for immunohistochemistry on placental sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry, and HTRA-4 is predominantly expressed in placental tissues where it localizes to the extracellular matrix and trophoblast compartments. For paraffin-embedded sections, antigen retrieval is typically required; citrate buffer at pH 6.0 with heat-induced epitope retrieval works well for serine proteases. Start with a 1:100 to 1:200 dilution and optimize based on signal intensity and background. Term placenta serves as an excellent positive control, while non-placental tissues such as liver or spleen provide useful negative controls for specificity.

Will this rabbit polyclonal HTRA-4 antibody cross-react with mouse or rat samples?

Human HTRA-4 is validated with this antibody. Mouse and rat cross-reactivity are predicted based on sequence homology but not experimentally confirmed. Human HTRA-4 shares approximately 75-80 percent identity with rodent orthologs, particularly in the catalytic serine protease domain, so cross-reactivity is plausible. If you are working with mouse placenta or rat trophoblast models, we recommend empirical testing at 1:500 to 1:1000 with appropriate positive controls. Non-human primate samples, especially from macaque placenta, are more likely to show robust cross-reactivity given higher sequence conservation with human HTRA-4.

What are good positive and negative controls for HTRA-4 expression studies?

Term human placenta is the gold-standard positive control, as HTRA-4 is predominantly expressed in trophoblast and decidual tissues. First-trimester placental villi also express HTRA-4 during invasion and remodeling. For cell lines, transfected HEK293 or trophoblast cell lines such as HTR-8/SVneo or JEG-3 serve as useful models, though endogenous expression varies. Negative controls should include tissues with minimal or absent HTRA-4 expression such as liver, brain, or skeletal muscle. For knockout validation, HTRA-4 siRNA-treated cells provide a functional negative control to confirm antibody specificity and rule out off-target binding.

How should I prepare samples to detect secreted HTRA-4 in conditioned media?

Because HTRA-4 is a secreted protease, conditioned media from cultured placental explants or trophoblast cell lines are appropriate sample sources. Collect serum-free or low-serum media after 24-48 hours of conditioning, then concentrate by ultrafiltration or TCA precipitation to enrich secreted proteins. Aim for 10-20 micrograms total protein per lane. Include protease inhibitors during collection to prevent autodegradation, though be aware that HTRA-4 itself may cleave substrates in the extracellular space. For extracellular matrix fractions, sequential extraction with detergents or chaotropic agents may be required to solubilize matrix-associated HTRA-4.

What is the recommended storage condition for this HTRA-4 polyclonal antibody?

Store the antibody at minus twenty degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied at 100 micrograms and remains stable for at least twelve months under these conditions. For frequent use, a working aliquot can be kept at four degrees Celsius for up to one month with addition of 0.02 percent sodium azide as preservative. Avoid prolonged exposure to room temperature. If you observe precipitates after thawing, centrifuge briefly at 10,000 g before use to remove aggregates that may increase background signal.

Does HTRA-4 have isoforms or splice variants I should be aware of?

The primary annotated isoform of HTRA-4 is the 476-amino acid full-length protein. Alternative splice variants have been reported in the literature, but functional characterization remains limited and tissue-specific expression patterns are not well-defined. This polyclonal antibody was raised against a large immunogen spanning residues corresponding to most of the mature protein, so it should recognize the predominant isoform and potentially detect closely related variants if they retain shared epitopes. If you suspect multiple bands on Western blot, consider that proteolytic processing, glycosylation heterogeneity, or post-translational cleavage in the extracellular space may contribute to apparent size differences.