Anti-Serpin-B2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-B2 on a Western blot?

Serpin-B2 (PAI-2) migrates at approximately 47 kDa on SDS-PAGE under reducing conditions, corresponding to its 415-amino-acid sequence. You may observe a glycosylated form near 60 kDa if working with secreted Serpin-B2, though the predominant cytoplasmic isoform is non-glycosylated. Some cell types express both forms simultaneously. If you see a doublet or higher-molecular-weight species, confirm your sample source and consider whether your cells are stimulated or transformed, as glycosylation increases during differentiation or inflammatory activation.

Is this Serpin-B2 antibody suitable for detecting PAI-2 in monocyte or macrophage lysates?

Yes. Serpin-B2 is expressed at high levels in monocytes and macrophages, particularly upon LPS or cytokine stimulation. We recommend starting at 1:1000 dilution for Western blot of whole-cell lysates from THP-1, U937, or primary human monocytes. Basal expression can be low in unstimulated cells; consider treating with LPS, TNF-α, or PMA for 6 to 24 hours to induce robust Serpin-B2 signal. Placental tissue serves as a strong positive control.

Can I use this antibody to distinguish Serpin-B2 from PAI-1 in the same sample?

Yes. Despite functional overlap, Serpin-B2 and PAI-1 (Serpin-E1) are distinct gene products with no significant sequence homology outside the serpin domain. This polyclonal was raised against a Serpin-B2-specific epitope and does not cross-react with PAI-1 in our validation panels. On Western blot, PAI-1 migrates near 45 to 50 kDa, overlapping with non-glycosylated Serpin-B2, so molecular weight alone will not resolve them. Use isoform-specific antibodies or verify by siRNA knockdown if both serpins are abundant in your system.

What starting dilution should I use for immunohistochemistry on formalin-fixed tissue?

We recommend a starting dilution of 1:200 to 1:500 for IHC on FFPE sections, with citrate-based antigen retrieval at pH 6.0. Serpin-B2 staining is typically cytoplasmic in monocytes, macrophages, and syncytiotrophoblasts. For frozen sections, 1:500 to 1:1000 often works well. Titre empirically against your tissue type; expression is inducible and heterogeneous. Positive-control tissues include term placenta and inflamed colon or synovium. Normal liver and kidney are generally negative or very weakly positive.

Will this antibody detect Serpin-B2 in mouse or rat samples?

Cross-reactivity with rodent Serpin-B2 is predicted based on sequence homology but not formally validated. Human and mouse SERPINB2 share approximately 60 percent identity at the amino-acid level. If you are working with mouse models, we suggest testing at the recommended 1:1000 dilution and including a positive control such as LPS-stimulated mouse peritoneal macrophages. Monkey (non-human primate) reactivity is more likely given higher sequence conservation, but empirical validation remains advisable.

How should I prepare cell lysates to preserve Serpin-B2 signal for Western blot?

Serpin-B2 is a cytoplasmic protein and readily extracted in standard RIPA or NP-40 lysis buffers supplemented with protease inhibitors. Avoid prolonged incubation at room temperature; serpins can undergo conformational changes or proteolysis. Snap-freeze pellets or lyse on ice, then clarify at 4 degrees Celsius. Load 20 to 40 micrograms total protein per lane for most adherent cell lines; you may need more for weakly expressing primary cells. Boil samples in reducing Laemmli buffer for five minutes before loading.

What are recommended positive and negative control cell lines for Serpin-B2 expression?

THP-1 or U937 human monocytic leukemia cells, especially after PMA or LPS treatment, serve as robust positive controls. HT-1080 fibrosarcoma cells also express Serpin-B2 constitutively. For negative controls, HEK293T and NIH-3T3 typically show minimal or undetectable expression under basal conditions. Primary human keratinocytes and some epithelial lines express Serpin-B2 variably depending on passage and differentiation state. Placental villous tissue remains the gold-standard positive control for method development.

How stable is this antibody during repeated freeze-thaw cycles and what storage is recommended?

Store the antibody at minus 20 degrees Celsius as supplied. For routine use, aliquot upon receipt to minimize freeze-thaw cycles; rabbit polyclonal antibodies generally tolerate two to three cycles without major titre loss, but repeated thawing accelerates aggregation and can reduce signal. For long-term storage beyond one year, keep at minus 80 degrees Celsius. Avoid storing diluted antibody; prepare working dilutions fresh in blocking buffer on the day of use to maintain consistent performance across experiments.