Anti-HGF Activator Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for HGF Activator on Western blot?

HGF Activator is synthesized as a 655-amino acid zymogen with a predicted molecular weight around 70-80 kDa depending on glycosylation. On reducing Western blots you should observe the full-length proenzyme near 80 kDa in most lysates. Following proteolytic activation, HGFAC is cleaved into a heavy chain (approximately 50 kDa) and a catalytic light chain (approximately 30 kDa) connected by disulfide bonds. Under reducing conditions, you may see both chains if your sample contains activated enzyme. Plasma samples or conditioned media from injury models often show both forms. Start with 1:1000 dilution and adjust exposure time to visualize less abundant activated species.

Is this HGF Activator antibody specific or does it cross-react with other trypsin-like proteases?

This polyclonal antibody was raised against human HGF Activator and has been validated for specificity against the target. The trypsin-like serine protease family shares structural homology, so we recommend including appropriate controls—lysates from HGFAC-knockout cells or RNAi knockdown samples—to confirm band identity in your system. The antibody recognizes human HGFAC with validated reactivity and shows potential cross-reactivity with monkey orthologs based on sequence homology. We have not observed non-specific binding to closely related proteases like matriptase or hepsin in our validation work, but tissue-specific expression patterns should be confirmed with negative controls in your experimental context.

Can I use this antibody to detect HGF Activator in monkey or mouse samples?

The antibody has validated reactivity with human HGF Activator and predicted cross-reactivity with monkey (non-human primate) samples based on high sequence conservation in the immunogen region. Mouse HGFAC shares lower homology with human, and we have not validated reactivity in mouse lysates. If you are working with primate samples, start with the recommended 1:1000 dilution for Western blot. For mouse experiments, we suggest testing the antibody alongside a positive human control, but you may need a mouse-specific reagent. Always include species-matched recombinant protein or validated cell lysates as positive controls when working outside the validated species range.

What is the biological role of HGF Activator and why does it matter for my experiment?

HGF Activator is the principal serine protease responsible for converting inactive pro-HGF into its bioactive heterodimeric form, which then binds and activates the MET receptor. This proteolytic step is the rate-limiting trigger for HGF-MET signaling, which drives cell migration, proliferation, and survival in contexts ranging from wound healing to cancer invasion. HGFAC activity is spatially restricted in tissues, so its expression and activation state determine where and when HGF signaling occurs. If you are studying MET pathway activation, tumor microenvironment dynamics, tissue regeneration, or fibrosis, measuring HGFAC expression and processing gives insight into the regulatory control upstream of receptor engagement. Dysregulation is linked to metastasis and aberrant repair.

What sample preparation steps improve HGF Activator detection in Western blot or IHC?

For Western blot, use RIPA or similar lysis buffer with protease inhibitors to preserve both the zymogen and activated forms. HGFAC is secreted, so conditioned media, serum, or plasma samples are rich sources; concentrate media by ultrafiltration or precipitation if necessary. Avoid over-boiling samples if you want to assess the disulfide-linked heterodimer under non-reducing conditions. For IHC, HGFAC is found in extracellular matrix and stromal compartments in liver, lung, and tumor tissues. Antigen retrieval with citrate buffer (pH 6.0) typically works well for formalin-fixed paraffin-embedded sections. Include positive-control tissues known to express HGFAC, such as liver or wound-healing models, to optimize staining conditions.

What dilution should I start with for Western blot and how do I optimize it?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this antibody. Optimal dilution depends on HGFAC abundance in your sample—expression is generally low in resting cells but upregulated during injury, inflammation, or in certain tumors. If your signal is weak, try 1:500 or increase protein load to 30-50 µg per lane. If background is high, extend blocking time to two hours with 5 percent non-fat milk in TBST and dilute primary antibody to 1:2000. Include a positive control lysate expressing known levels of HGFAC to benchmark sensitivity and a negative control to assess specificity across your titration series.

How should I store the HGF Activator antibody and what is the expected shelf life?

Store the antibody at –20°C upon arrival. For routine use, prepare single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce antibody titer and increase aggregation. Aliquots can be stored at –20°C for at least 12 months under these conditions. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month in the presence of 0.02 percent sodium azide as preservative, though we recommend frozen storage for maximum long-term stability. Always centrifuge thawed aliquots briefly before use to pellet any aggregates. Do not store diluted antibody in blocking buffer for more than one week even at 4°C, as this reduces performance.

What controls should I include when validating HGF Activator expression in my system?

Include a positive control known to express HGFAC, such as HepG2 liver cells, primary hepatocytes, or conditioned medium from fibroblasts stimulated with pro-inflammatory cytokines, which upregulate protease expression. A negative control is equally critical—use lysate from cells with CRISPR knockout or stable shRNA knockdown of HGFAC, or a tissue known to lack expression. For functional validation, treat samples with recombinant HGFAC or compare pro-HGF activation status in parallel. Blocking-peptide competition is useful for polyclonal antibodies; pre-incubate antibody with excess immunogen peptide to confirm that the signal disappears. These controls distinguish specific signal from cross-reactive or non-specific bands in complex lysates.