Anti-HAI-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for HAI-2 on a Western blot?

HAI-2 (SPINT2) is a 252-residue type I transmembrane protein with a predicted molecular weight around 27 kDa from its amino acid sequence. However, on Western blot you will typically observe a band near 30-35 kDa due to post-translational glycosylation at multiple N-glycosylation sites in the extracellular Kunitz domains. Under reducing conditions, you should see a single band corresponding to the full-length membrane-anchored form. If you observe higher molecular weight species, consider that HAI-2 can form complexes with its target proteases such as matriptase, though these are usually dissociated under standard denaturing SDS-PAGE conditions.

What starting dilution should I use for HAI-2 Western blot experiments?

For this rabbit polyclonal HAI-2 antibody, the recommended starting dilution for Western blot is 1:1000 in blocking buffer containing 5 percent non-fat milk or BSA in TBST. This dilution has been validated against human lysates. Depending on HAI-2 expression levels in your sample type and your detection system sensitivity, you may need to optimize between 1:500 and 1:2000. Epithelial tissues and cell lines such as Caco-2, MDCK, or MCF-7 typically express readily detectable levels of HAI-2 and serve as good positive controls for initial titration experiments.

Does this HAI-2 antibody cross-react with HAI-1 or other Kunitz domain proteins?

HAI-2 shares structural similarity with HAI-1 (SPINT1), as both are membrane-bound Kunitz-type serine protease inhibitors, but they are distinct gene products with approximately 40 percent sequence identity. This polyclonal antibody was raised against a HAI-2-specific immunogen and shows no detectable cross-reactivity with HAI-1 in our validation studies. However, because it is a polyclonal reagent recognizing multiple epitopes across the HAI-2 extracellular region, we recommend including HAI-1-overexpressing lysates or SPINT2 knockout controls in your initial experiments if you work with samples where both inhibitors are co-expressed at high levels.

Will this antibody detect HAI-2 in non-human species like mouse or rat?

This antibody has been validated for reactivity with human HAI-2. Cross-reactivity with monkey and dog is predicted based on high sequence conservation in the immunogen region across these species, but has not been experimentally validated. Mouse and rat HAI-2 share approximately 70-75 percent identity with human, so cross-reactivity is possible but not guaranteed. If you are working with rodent samples, we recommend running a side-by-side comparison with human lysate as a positive control in your first experiment. Alternatively, consider including a species-matched positive control tissue such as mouse intestinal epithelium, where HAI-2 is abundantly expressed.

What positive control samples should I use to validate HAI-2 detection?

HAI-2 is broadly expressed in epithelial tissues, so human placenta, kidney cortex, or colon epithelium lysates serve as excellent positive controls. Among common cell lines, Caco-2 (colorectal adenocarcinoma), MDCK (canine kidney), and HaCaT (keratinocytes) express detectable levels of endogenous HAI-2. If you are studying HAI-2 regulation or protease co-localization, consider including lysates treated with phorbol esters or inflammatory cytokines, which can modulate HAI-2 expression. For IHC validation, normal human placenta or kidney sections provide robust signal at the apical surface of polarized epithelia where HAI-2 physiologically localizes.

Can I use this antibody for immunofluorescence on live or fixed cells?

Because HAI-2 is a type I transmembrane protein with its Kunitz domains projecting extracellularly, this antibody can potentially detect surface HAI-2 on non-permeabilized cells if the epitope is accessible. However, our validation data focuses on Western blot and IHC applications. For immunofluorescence, start with methanol-fixed or paraformaldehyde-fixed permeabilized cells at a 1:100 to 1:200 dilution. If you want to detect surface HAI-2 on live or non-permeabilized cells, perform a small-scale test first, as epitope accessibility depends on antibody binding site and cell-surface topology. Expect punctate or membrane-enriched staining at cell-cell junctions in polarized epithelia.

How should I store this HAI-2 antibody and what is the shelf life?

This rabbit polyclonal antibody is supplied as 100 µg in solution. Store at minus 20 degrees Celsius for long-term stability. For routine use, aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregate formation. An aliquot thawed for daily use can be kept at 4 degrees Celsius for up to one month if sodium azide is present as a preservative. Under proper storage at minus 20 degrees Celsius, polyclonal antibodies typically retain activity for at least two years from the date of manufacture. If you notice elevated background or reduced signal over time, compare performance against a freshly thawed aliquot before troubleshooting other variables.

Does HAI-2 exist as multiple isoforms or processed fragments I should know about?

HAI-2 is encoded by the SPINT2 gene as a single-pass transmembrane protein. While alternative splicing has been reported in the literature, the predominant form is the full-length 252-residue isoform containing two tandem Kunitz domains (KD1 and KD2) and a transmembrane anchor. You may occasionally detect a soluble form of HAI-2 in conditioned media or serum, arising from ectodomain shedding by metalloproteases or other sheddases. On Western blot, this shed form migrates slightly faster than the full-length protein. If you are analyzing secreted fractions, include protease inhibitors during sample preparation to minimize artifactual cleavage and preserve the endogenous shedding pattern.