Anti-Serpin-B12 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Serpin-B12 on Western blot?

Serpin-B12 runs at approximately 46 kDa on reducing SDS-PAGE, corresponding to the 405-amino acid full-length protein. Because Serpin-B12 lacks a signal peptide and is not glycosylated or heavily post-translationally modified, the observed band typically matches the predicted molecular weight closely. Occasionally, researchers report minor shifts due to phosphorylation or sample preparation artifacts. If you see multiple bands, consider the possibility of partial degradation in lysates stored without protease inhibitors, or cross-reactivity with related clade B serpins in overloaded samples.

Does this Serpin-B12 antibody cross-react with other serpin family members?

The polyclonal was raised against a region spanning residues 46–460 of human Serpin-B12, which includes sequences divergent from closely related clade B serpins such as Serpin-B2 and Serpin-B6. Cross-reactivity with other serpin family members has not been systematically ruled out but is unlikely at the recommended 1:1000 dilution for Western blot. If you work with cell lines or tissues expressing multiple clade B serpins at high levels, include a Serpin-B12 knockdown or knockout lysate as a specificity control to confirm the band corresponds to your target.

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

For Western blot, start at 1:1000 in 5% non-fat dry milk or BSA in TBST; this dilution has been validated with whole-cell lysates from human cell lines. For immunohistochemistry on formalin-fixed paraffin-embedded tissue, a typical starting range is 1:200 to 1:500 after heat-induced epitope retrieval in citrate buffer, pH 6.0. Because Serpin-B12 is cytoplasmic, expect diffuse cytoplasmic staining rather than membrane or nuclear signal. Titrate the antibody against your specific sample type and detection system to minimize background while preserving signal.

Is Serpin-B12 expression tissue-specific, and what are good positive control lysates?

Serpin-B12 is expressed in multiple tissues, with detectable levels in liver, kidney, and certain epithelial cell types. Expression levels vary and may be upregulated during differentiation or in response to specific stimuli. For a reliable positive control in Western blot, use lysates from HepG2 or HEK293 cells, which typically express endogenous Serpin-B12. Alternatively, transiently transfect cells with a Serpin-B12 expression construct to generate a high-signal control. Avoid assuming uniform expression across all cell lines without prior validation, especially in immortalized or transformed contexts.

Will this rabbit polyclonal Serpin-B12 antibody work in non-human primate samples?

The antibody is validated for human samples and predicted to cross-react with Old World monkey orthologs, given the high sequence conservation of Serpin-B12 across primates. Cynomolgus and rhesus macaque Serpin-B12 share greater than 95% identity with the human sequence in the immunogen region. However, cross-reactivity has not been formally validated, so we recommend testing a dilution series on monkey lysates and including a human positive control in parallel. Reactivity with New World monkeys or more distantly related species is less predictable and should be confirmed experimentally.

Does Serpin-B12 require reducing conditions for Western blot detection?

Serpin-B12 is a cytoplasmic protein and does not contain disulfide bonds critical for epitope accessibility, so reducing conditions are not strictly required for detection by this polyclonal antibody. Standard reducing sample buffer with DTT or beta-mercaptoethanol at 95–100°C for 5 minutes works well and is recommended for reproducibility. Non-reducing conditions are also compatible if you are examining complex formation with target proteases, but expect the same 46 kDa band unless Serpin-B12 is covalently cross-linked to a protease in a stable inhibitory complex.

What is the recommended storage condition and shelf life for this Serpin-B12 antibody?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied in a glycerol-containing buffer that remains liquid at –20°C, allowing direct pipetting without thawing if you prefer. Under these conditions, expect stable performance for at least 12 months from receipt. For long-term storage beyond one year, keep at –80°C. Avoid prolonged exposure to room temperature and do not store diluted antibody in working solution for more than one week at 4°C, as bacterial contamination and degradation can occur.

Can I use this antibody to detect Serpin-B12 secreted into cell culture media?

Serpin-B12 lacks a signal peptide and is localized to the cytoplasm under normal physiological conditions, so it is not actively secreted. Detection in cell culture supernatants typically indicates cell lysis, necrosis, or passive release from damaged cells rather than bona fide secretion. If you detect Serpin-B12 in conditioned media, check for lactate dehydrogenase or other cytoplasmic markers to assess membrane integrity. For experiments specifically examining intracellular protease regulation, focus on whole-cell lysates or cytoplasmic fractions rather than culture supernatants to capture the biologically relevant pool of Serpin-B12.