Anti-Serpin-B5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-B5 on a Western blot?

Serpin-B5 migrates at approximately 42 kDa on reducing SDS-PAGE, which corresponds to the 375-amino acid full-length protein. Some laboratories report a band closer to 45 kDa depending on gel percentage and running conditions. The protein lacks a signal peptide despite being secreted, so you will not observe higher-molecular-weight precursor forms. If you detect multiple bands, consider post-translational modifications or proteolytic fragments, particularly in tumor lysates where Serpin-B5 processing has been reported. Start with 1:1000 dilution in 5% milk or BSA and optimize exposure times to avoid overexposure of high-expressing normal epithelial samples.

Does this antibody recognize mouse or rat Serpin-B5 for cross-species studies?

This antibody is validated for human Serpin-B5. Mouse and rat orthologs share approximately 60-65% sequence identity with human SERPINB5, so cross-reactivity is not guaranteed and has not been experimentally validated by us. If you require detection in rodent models, we recommend running parallel positive controls with human tissue or recombinant protein and testing lysates from mouse mammary epithelium or prostate, where murine Serpinb5 is expressed. Empirical testing at 1:500 to 1:1000 dilution is the only reliable way to assess cross-reactivity. Predicted epitopes suggest limited conservation in the immunogenic regions.

What are good positive and negative control tissues for Serpin-B5 expression?

Normal mammary epithelium, prostate epithelium, and skin keratinocytes are strong positive controls for Serpin-B5 expression. MCF-7 breast cancer cells retain Serpin-B5 expression and serve as a convenient positive lysate. For negative controls, use HeLa cervical carcinoma or MDA-MB-231 triple-negative breast cancer cells, which commonly lose Serpin-B5 during malignant transformation. When interpreting tumor samples, be aware that Serpin-B5 downregulation is a frequent event in progression, so absence of signal may reflect biology rather than technical failure. Always include a positive control lane to confirm antibody performance and transfer efficiency in each experiment.

Can I use this Serpin-B5 antibody for immunohistochemistry on FFPE sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry, including paraffin-embedded tissue. Serpin-B5 is predominantly cytoplasmic with some extracellular localization, so expect diffuse cytoplasmic staining in normal epithelium. Antigen retrieval is typically required; citrate buffer pH 6.0 with heat-induced epitope retrieval works well for most serpin family members. Start with a 1:100 to 1:200 dilution and titrate based on background. Nuclear staining occasionally appears in some tumor contexts but remains controversial in the literature. Use normal mammary or prostate tissue as your positive control and include a no-primary-antibody section to assess nonspecific binding of your secondary detection system.

Does Serpin-B5 function as a protease inhibitor like other serpins?

No, Serpin-B5 is an atypical serpin that lacks canonical serine protease inhibitory activity. The reactive center loop and hinge region do not undergo the stressed-to-relaxed conformational change required for protease trapping. Instead, Serpin-B5 functions as a tumor suppressor through protease-independent mechanisms, including modulation of cell adhesion, migration, and extracellular matrix interactions. When studying Serpin-B5, do not expect protease inhibition assays to yield activity. Its tumor-suppressive effects are mediated by binding partners such as beta-1 integrin and collagen, and by influencing cell surface receptor signaling. This makes functional validation quite different from classical serpin family members.

What dilution should I start with for Western blot with this antibody?

We recommend starting at 1:1000 dilution for Western blot with this rabbit polyclonal Serpin-B5 antibody. This ratio typically provides strong signal in lysates from Serpin-B5-expressing cells such as MCF-7 or normal mammary epithelium, with 20-30 micrograms total protein per lane. Incubate primary antibody overnight at 4°C in either 5% non-fat milk or 3-5% BSA in TBST; BSA may reduce background if you observe high nonspecific binding. If your signal is weak in tumor samples, remember that Serpin-B5 is frequently downregulated during malignant transformation, so absence of band may be biological. You can try 1:500 dilution, but verify specificity with positive control lysate in parallel.

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

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100-microgram pack size is suitable for aliquoting into 10-20 microliter volumes depending on your typical experiment frequency. Rabbit polyclonal antibodies in glycerol-containing storage buffer generally remain stable for at least two years at -20°C when handled properly. Avoid prolonged exposure to room temperature; thaw on ice and return to freezer promptly. If you observe increased background or decreased signal over time, it likely reflects freeze-thaw damage rather than antibody degradation. For long-term projects, prepare sufficient aliquots at the outset to maintain consistency across experiments.

Why might I see loss of Serpin-B5 signal in my tumor samples?

Loss of Serpin-B5 expression is a well-documented event during malignant transformation of mammary, prostate, and other epithelial tumors. Downregulation occurs through promoter hypermethylation, loss of heterozygosity, or transcriptional repression, and correlates with increased metastatic potential and poor prognosis in multiple cancer types. If you detect Serpin-B5 in normal adjacent tissue but not tumor, this likely reflects biological silencing rather than technical failure. Always run a positive control such as MCF-7 lysate or normal epithelium on the same blot or slide to confirm antibody performance. Conversely, re-expression of Serpin-B5 in some aggressive tumor subsets has been reported, so context matters when interpreting staining patterns.