Anti-HTRA-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for HTRA-1 on Western blot?

The full-length HTRA-1 protein runs at approximately 50 kDa on reducing SDS-PAGE, corresponding to the 480-residue polypeptide plus glycosylation. Because HTRA-1 is secreted and undergoes N-glycosylation, you may observe a smear or band between 48–55 kDa depending on cell type and post-translational modification status. In conditioned media samples, processed or proteolytically cleaved forms occasionally appear as lower-molecular-weight bands. Deglycosylation with PNGase F will shift the band to approximately 48 kDa, confirming the glycosylated nature of the mature protein.

What starting dilution should I use for Western blot with this HTRA-1 antibody?

The recommended starting dilution is 1:1000 in blocking buffer for Western blot. This polyclonal antibody typically yields clean bands at that concentration when detecting HTRA-1 from whole-cell lysates or conditioned media. If your sample has low HTRA-1 expression or you are working with tissue lysates, start at 1:500 and titrate up to 1:2000 to balance signal intensity and background. Overnight incubation at 4°C often improves sensitivity compared to one-hour room-temperature binding. Always include a positive control lysate from RPE or fibroblast cells, which express HTRA-1 constitutively.

Does this antibody detect mouse and rat HTRA-1, or only human?

This antibody has been validated for human HTRA-1 and shows predicted cross-reactivity with mouse, rat, non-human primate, and Pan troglodytes based on sequence homology in the immunogen region. Human and mouse HTRA-1 share approximately 87% identity, so cross-reactivity is likely but should be confirmed in your hands with appropriate species-matched positive controls. If you plan to use mouse or rat samples, run a pilot blot at 1:500–1:1000 and compare signal to a validated human control. We have not performed formal validation in rodent models, so treat preliminary results as predicted until confirmed.

Can I use this HTRA-1 antibody for immunohistochemistry or immunofluorescence?

Yes, Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry, and many perform well in immunofluorescence. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval with citrate buffer (pH 6.0) at high temperature is typically required because HTRA-1 epitopes are masked by fixation. Start with a 1:100–1:200 dilution and optimize based on signal and background. For IF on cultured cells, try 1:100 after fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100. HTRA-1 localizes to secretory vesicles and the extracellular matrix in many cell types.

What sample types work best for detecting HTRA-1 with this antibody?

HTRA-1 is a secreted protease, so conditioned serum-free media from cultured cells often yield the strongest signal on Western blot. Collect media after 24–48 hours, concentrate 10–20-fold using centrifugal filters (10 kDa cutoff), and load 10–20 µg total protein per lane. Whole-cell lysates also work well, particularly from retinal pigment epithelial cells, fibroblasts, or osteoarthritic chondrocytes, which express HTRA-1 at moderate-to-high levels. Tissue lysates from placenta, skeletal muscle, or cartilage are suitable but may require more total protein. Use RIPA buffer with protease inhibitors; avoid boiling samples if studying oligomeric forms.

How should HTRA-1 antibody aliquots be stored to maintain long-term stability?

Store the antibody at –20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. After the first thaw, the working aliquot can be kept at 4°C for up to one month if sodium azide (0.02–0.05%) is present as a preservative. Do not store diluted antibody in blocking buffer for more than one week; prepare fresh dilutions for critical experiments. If you observe a gradual loss of signal over time, it usually reflects freeze-thaw damage rather than lot-to-lot variation. Glycerol (50%) can be added to the stock for –20°C storage without freezing solid.

What controls should I include when detecting HTRA-1 in cell lysates or tissue?

Include a positive control lysate from ARPE-19 retinal pigment epithelial cells or primary human fibroblasts, both of which express HTRA-1 constitutively. For a negative control, use lysate from cells treated with HTRA-1-targeting siRNA or CRISPR knockout lines if available. Loading equal amounts of total protein (20–30 µg per lane) and probing for a housekeeping protein such as GAPDH or β-actin on the same membrane confirms equal loading. If studying extracellular HTRA-1, run unconditioned serum-free medium as a blank to rule out serum contamination. A no-primary-antibody control verifies that secondary antibody does not bind nonspecifically.

Does HTRA-1 have splice isoforms I should be aware of on Western blot?

HTRA-1 is encoded by a single-exon open reading frame with no well-characterized splice isoforms that produce stable, functional proteins. You should see one predominant band at approximately 50 kDa representing the mature secreted form. However, intracellular precursor forms with the signal peptide intact may run slightly higher, around 51–52 kDa, if your lysate captures nascent protein before cleavage. Polymorphisms in the promoter region (such as rs11200638) affect expression levels rather than protein size. If you observe multiple bands, consider proteolytic processing, post-translational modification heterogeneity, or degradation during sample preparation rather than alternative splicing.