Anti-HTRA-3 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for HTRA-3 on a Western blot?

The full-length HTRA-3 protein is 453 amino acids with a predicted molecular weight around 48-50 kDa. However, HTRA-3 is a secreted protease with an N-terminal signal peptide that is cleaved during maturation. The mature, secreted form typically migrates at approximately 45-47 kDa on reducing SDS-PAGE. You may also observe higher molecular weight species corresponding to glycosylated forms or processing intermediates, depending on cell type and secretion status. For intracellular lysates, expect the mature form; for conditioned media from secreting cells, the band should be prominent in the 45-47 kDa range.

What dilution should I start with for Western blot detection of HTRA-3?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody. This dilution has been validated with human samples and typically yields good signal-to-noise ratios with standard chemiluminescent detection. For low-abundance samples or conditioned media where HTRA-3 may be dilute, you can titrate up to 1:500. Conversely, if background is high or your lysate is particularly rich in HTRA-3, try 1:2000. Optimal dilution depends on expression level, sample type, and detection system sensitivity, so empirical optimization against your specific lysate is advisable.

Does HTRA-3 function require specific activation or cofactors I should consider?

HTRA-3 is a serine protease that exhibits temperature-dependent and substrate-dependent activation, typical of HtrA family members. The enzyme is synthesized as a zymogen and undergoes autocatalytic activation. Proteolytic activity is optimal at physiological pH and can be modulated by calcium and other divalent cations in some contexts. If you are studying HTRA-3 enzymatic activity rather than just protein expression, consider that its protease function may be influenced by temperature, ionic strength, and the presence of substrate proteins like decorin, biglycan, or fibronectin. For Western blot detection, these functional considerations do not affect antibody binding.

Will this antibody detect mouse or rat HTRA-3 in my samples?

This antibody has been validated for human HTRA-3 and shows potential cross-reactivity with mouse, rat, and other mammalian orthologs based on sequence homology. Human and mouse HTRA-3 share approximately 75-80 percent amino acid identity, with high conservation in the protease and PDZ domains. We predict cross-reactivity, but it has not been formally validated in our hands. If you are working with rodent samples, we recommend testing the antibody at the suggested 1:1000 dilution initially and including a positive control such as recombinant mouse HTRA-3 or lysate from a tissue known to express it, such as placenta or ovary.

What are appropriate positive and negative controls for HTRA-3 expression?

HTRA-3 is highly expressed in placental tissue, particularly in trophoblast cells, making human placental lysate an excellent positive control. Ovarian granulosa cells and luteal tissue also express HTRA-3 during differentiation. For cell lines, JAR choriocarcinoma cells or primary trophoblast cultures are good choices. As a secreted protein, conditioned media from expressing cells can serve as a positive control. For negative controls, many immortalized cell lines and non-reproductive tissues express little to no HTRA-3 under basal conditions. HEK293 or HeLa cells typically show low or absent expression, though this should be confirmed empirically for your culture conditions.

Can I use this HTRA-3 antibody for immunohistochemistry or immunofluorescence?

Triple Point Biologics antibodies are routinely validated for Western blot and immunohistochemistry applications, and this rabbit polyclonal is suitable for IHC and immunofluorescence. 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 reasonable starting point. For immunofluorescence, dilutions in the range of 1:100 to 1:500 are common, though this depends on fixation method and tissue permeability. Human placenta sections serve as an excellent positive control for protocol optimization. Empirical titration is recommended for your specific tissue and fixation conditions.

How should I prepare samples to detect secreted HTRA-3 in conditioned media?

HTRA-3 is a secreted protease, so conditioned media from cultured cells is a physiologically relevant sample type. Collect serum-free or low-serum media after 24-48 hours of conditioning to minimize background. Concentrate the media 10- to 20-fold using centrifugal filter units with a 10 kDa cutoff to enrich secreted proteins and improve detection sensitivity. Add protease inhibitors immediately upon collection if you are concerned about degradation, though HTRA-3 itself is relatively stable. Load 15-30 microliters of concentrated media per lane and expect the mature secreted form around 45-47 kDa. Include cell lysate as a reference for intracellular precursor forms.

What is the recommended storage condition and shelf life for this antibody?

This rabbit polyclonal antibody is supplied as a 100 microgram aliquot and should be stored at -20 degrees Celsius for long-term stability. For routine use, we recommend preparing small working aliquots to avoid repeated freeze-thaw cycles, which can reduce antibody titer and increase aggregation. An aliquot in use can be kept at 4 degrees Celsius for up to one month with sodium azide as preservative. Under proper storage at -20 degrees Celsius, the antibody remains stable for at least two years from the date of receipt. Avoid storing in frost-free freezers where temperature cycling can occur.