Anti-HAI-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for HAI-1 on Western blot?

HAI-1 migrates at approximately 58-66 kDa on Western blot, which is higher than the predicted molecular weight of the 529-residue polypeptide due to N-linked glycosylation. You may observe multiple bands representing differentially glycosylated forms or proteolytic fragments. Some cell types also produce a cleaved, secreted form of HAI-1 lacking the transmembrane domain, which migrates slightly faster. Reducing conditions are recommended to disrupt any disulfide-linked complexes with target proteases like matriptase, which can shift the apparent molecular weight upward.

What is the recommended starting dilution for HAI-1 antibody in Western blot?

Start at 1:1000 dilution in blocking buffer containing 5 percent non-fat milk or BSA for overnight incubation at 4°C. This dilution has been validated with whole-cell lysates from epithelial cell lines expressing endogenous HAI-1. If signal is weak in your specific lysate, you can increase concentration to 1:500. Conversely, high-expressing samples may tolerate 1:2000. Always include a positive control lysate from a HAI-1-expressing epithelial cell line such as HaCaT or Caco-2 to confirm antibody performance before troubleshooting.

Does this antibody recognize HAI-1 in mouse and rat samples?

This antibody has validated reactivity against human HAI-1. Cross-reactivity with mouse and rat HAI-1 is predicted based on sequence homology in the immunogen region but has not been experimentally validated by Triple Point Biologics. Human and mouse HAI-1 share approximately 80 percent sequence identity. If you are working with rodent samples, plan to include a positive control from mouse or rat epithelial tissue and be prepared to optimize dilution. Some researchers report successful detection in mouse samples at similar working dilutions to human.

What are good positive and negative control cell lines for HAI-1 expression?

HAI-1 is widely expressed in epithelial cells, so keratinocytes (HaCaT), intestinal epithelial cells (Caco-2, HT-29), and prostate epithelial lines (LNCaP, PC-3) serve as reliable positive controls. Hepatocyte-derived cell lines like HepG2 also express moderate to high levels. For negative controls, consider mesenchymal or hematopoietic cells, though truly HAI-1-null lines are uncommon given its broad epithelial expression pattern. Alternatively, use siRNA knockdown in a positive cell line to confirm antibody specificity and establish the expected band shift on your blot.

Can this antibody detect HAI-1 in immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry. This rabbit polyclonal should perform well in IHC on formalin-fixed paraffin-embedded tissue sections; antigen retrieval with citrate buffer (pH 6.0) is typically effective for HAI-1. For immunofluorescence on cultured cells, start with a 1:100 to 1:200 dilution after fixation with 4 percent paraformaldehyde and permeabilization with 0.1 percent Triton X-100. HAI-1 localizes to the plasma membrane and cytoplasm in epithelial cells, so expect punctate membrane staining with some intracellular signal.

Does HAI-1 form complexes with proteases that affect antibody detection?

Yes. HAI-1 forms tight, SDS-stable complexes with its target serine proteases, particularly matriptase (ST14) and HGFAC. Under non-reducing conditions, you may detect high-molecular-weight bands corresponding to HAI-1-protease complexes in addition to the free HAI-1 monomer. If you are specifically interested in the free inhibitor pool, run samples under reducing conditions with dithiothreitol or beta-mercaptoethanol to disrupt disulfide linkages. Conversely, non-reducing gels can provide insight into the extent of protease inhibition and complex formation in your experimental system.

How should I store the HAI-1 antibody and how long is it stable?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase background. The antibody is supplied at 100 µg in a glycerol-containing buffer that remains stable for at least 12 months at -20°C. For frequent use, keep a working aliquot at 4°C with 0.02 percent sodium azide; this remains stable for several weeks. Avoid prolonged storage at 4°C without preservative. Do not store diluted antibody in blocking buffer for more than one week, as carrier proteins can degrade and increase non-specific binding.

Why is HAI-1 expression important in epithelial cancer research?

HAI-1 acts as a tumor suppressor in many epithelial cancers by inhibiting matriptase and hepatocyte growth factor activator, proteases that promote invasive growth and metastasis. Loss or downregulation of HAI-1 is associated with increased tumor aggressiveness in gastric, colorectal, ovarian, and prostate cancers. Conversely, maintaining HAI-1 expression preserves epithelial barrier integrity and restrains protease-driven signaling cascades. When designing cancer cell experiments, monitor HAI-1 levels alongside its target proteases to understand the balance of proteolytic regulation. Dysregulated HAI-1 can serve as a prognostic marker in patient-derived samples.