Anti-Serpin-B8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-B8 on a Western blot?

Serpin-B8 (PI-8) migrates at approximately 42–45 kDa on reducing SDS-PAGE, consistent with its 374-residue predicted mass of roughly 42.8 kDa. Because Serpin-B8 is an intracellular serpin lacking a signal peptide and significant post-translational glycosylation, the observed band usually tracks close to the calculated molecular weight. Minor gel-shift variability can occur depending on lysate oxidation state or protease cleavage during sample preparation. Using fresh lysate with a cocktail of protease inhibitors will yield the sharpest band at the expected size.

Which tissue or cell lysates serve as good positive controls for Serpin-B8?

Serpin-B8 is broadly expressed in epithelial tissues, with particularly strong signal reported in skin keratinocytes, placenta, and stratified squamous epithelia. For validation, human keratinocyte lines such as HaCaT or primary epidermal keratinocyte lysates are reliable positive controls. Placental tissue lysate is another high-expressing source. Non-epithelial lines such as lymphoid or fibroblast cultures typically show lower or undetectable Serpin-B8, making them useful negative or low-expression comparators. Confirm expression in your specific cell model by RT-PCR or publicly available RNA-seq datasets before troubleshooting antibody performance.

What starting dilution should I use for Western blot with this Serpin-B8 antibody?

We recommend starting at 1:1000 dilution for Western blot, based on our internal validation in human epithelial lysates. This polyclonal rabbit antibody was raised against a broad immunogen spanning Serpin-B8 residues, so epitope accessibility is typically good under standard denaturing conditions. If your lysate has low Serpin-B8 expression or if background is minimal after the first trial, you can increase concentration to 1:500. Conversely, if non-specific bands appear, dilute further to 1:2000. Always include a no-primary control to distinguish true signal from secondary-antibody artifacts.

Does Serpin-B8 form complexes with proteases that change the Western blot pattern?

Yes. Serpin-B8 functions as a suicide-substrate inhibitor that forms stable, covalent SDS-resistant complexes with target serine proteases such as furin. These serpin–protease complexes migrate at higher apparent molecular weights—typically 80–120 kDa depending on the protease—and can appear as additional bands above the 42–45 kDa monomer. The ratio of monomer to complex varies with cellular protease activity and stress state. If you observe high-molecular-weight smearing or discrete upper bands, consider treating lysate with reducing agent and boiling to maximize monomer recovery, though some complexes remain stable even under denaturing conditions.

Is this Serpin-B8 antibody cross-reactive with mouse or rat samples?

Human reactivity is validated; mouse and rat cross-reactivity are predicted but not yet confirmed in-house. Serpin-B8 shares approximately 70–75 percent sequence identity between human and rodent orthologs, with conserved epitopes in the core serpin domain. Researchers have reported variable success using human-Serpin-B8 antibodies on mouse tissue, often requiring optimization of dilution and fixation for immunohistochemistry. We recommend pilot Western blots on mouse or rat epithelial lysates at 1:500 and 1:1000 to assess signal strength and specificity before committing to larger experiments. Include a validated human control in parallel.

Can I use this antibody for immunofluorescence on fixed keratinocytes?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence as well as Western blot and IHC. For Serpin-B8 immunofluorescence in cultured keratinocytes, fix cells in four percent paraformaldehyde, permeabilize with 0.1–0.2 percent Triton X-100, and start antibody dilution at 1:100 to 1:200. Because Serpin-B8 is cytoplasmic and enriched at desmosomes, you should observe diffuse cytosolic staining with focal concentration at cell–cell junctions. Co-staining with desmosomal markers such as desmoplakin can confirm localization. Optimize blocking and wash steps to minimize background in your specific fixation protocol.

How should I store this Serpin-B8 antibody and how long does it remain stable?

Store the antibody at –20 °C in small aliquots to avoid repeated freeze–thaw cycles, which can reduce titer and increase aggregation. The polyclonal is supplied in a stabilizing buffer; once thawed, an aliquot can be held at 4 °C for up to one month for routine use. Do not store diluted working solutions; prepare fresh in blocking buffer for each experiment. Under these conditions, the antibody retains reactivity for at least two years from receipt. If you observe declining signal or increased background over time, prepare a fresh aliquot and re-optimize dilution.

What is the biological role of Serpin-B8 and why does expression vary across my samples?

Serpin-B8 is an intracellular inhibitor of furin and related proprotein convertases, implicated in regulating desmosome-mediated cell adhesion and epithelial integrity. Expression is highest in stratified squamous epithelia—skin, oral mucosa, esophagus—and is inducible by differentiation signals, cytokines, and cellular stress. Variability across your samples likely reflects differences in epithelial differentiation state, confluence, or inflammatory milieu. For instance, subconfluent or proliferating keratinocytes may show lower Serpin-B8 than differentiated, contact-inhibited cultures. Normalizing culture conditions or tracking differentiation markers alongside Serpin-B8 can clarify sample-to-sample variation.