Anti-Serpin-B1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-B1 on a Western blot?

Native Serpin-B1 runs at approximately 42-45 kDa on reducing SDS-PAGE, consistent with the 379-residue mature protein (predicted ~42.7 kDa). In neutrophil and monocyte lysates the cleaved form (RCL-cleaved, ~38-40 kDa) is often visible as a lower band, particularly if samples were not fully protease-inhibited during lysis. SDS-stable covalent complexes with neutrophil elastase, cathepsin G, or proteinase-3 can also appear at ~70-95 kDa. If you see only high-MW smearing, suspect incomplete reduction or complex formation; boil 5 minutes in fresh DTT/β-ME-containing Laemmli buffer.

What dilution should I start with for Western blot, and what lysate loading works?

Start at 1:1000 in 5% non-fat milk or BSA/TBST, overnight at 4°C. Serpin-B1 is highly abundant in neutrophils, so 10-20 µg of whole-neutrophil lysate is usually sufficient; for monocytes, macrophages, or epithelial lines load 30-50 µg. For low-expressing tissues or transfected systems, titre between 1:500 and 1:2000 against your specific lysate. The 100 µg pack at 1:1000 supports roughly 100 mini-blots at 10 mL working volume. Milk works well; we have not seen phospho-epitope interference, so BSA is not required.

Does this antibody cross-react with other clade B serpins like SerpinB6 or SerpinB9?

The immunogen targets sequence within human SERPINB1 that shares limited identity with paralogues SerpinB6, B8, and B9 in the reactive centre loop and shutter region, but clade B serpins do share a conserved fold. We have not observed cross-reactivity with recombinant SerpinB6 or B9 on Western blot, but if you are working in tissues co-expressing multiple ov-serpins (e.g., spleen, bone marrow), include a SERPINB1-knockdown or knockout control. SerpinB1-/- mouse neutrophils are a clean negative control once mouse reactivity is confirmed in your hands.

Is this antibody validated for mouse or rat samples?

Validation to date is on human samples (neutrophil lysate, HL-60, monocyte preparations). Reactivity with mouse, rat, pan, monkey, dog, and pig is predicted based on sequence alignment of the immunogen region, but has not been formally validated in-house. Mouse Serpinb1a shares ~70% identity with human SERPINB1 across the immunogen; expect detection but titre your dilution and include a positive control (mouse bone marrow neutrophils run high). For rat, we suggest a pilot WB at 1:500 and 1:1000 before scaling to IHC.

What positive control should I use to confirm the antibody is working?

Human peripheral blood neutrophils are the gold-standard positive control — Serpin-B1 is among the most abundant cytoplasmic proteins, and 5-10 µg of lysate gives a strong 42-45 kDa band. HL-60 cells differentiated toward granulocytes with DMSO or ATRA also express robust Serpin-B1. U937 (monocytic) shows moderate expression. For tissue, human bone marrow, spleen, and lung (alveolar macrophage-rich) work well for IHC. Avoid using whole blood without leukocyte enrichment, as plasma serpins (SerpinA1, A3) dominate and can confuse interpretation.

How should I prepare neutrophil lysates to avoid Serpin-B1 degradation or complex artefacts?

Neutrophils carry abundant active serine proteases that will cleave Serpin-B1's reactive centre loop the moment cells lyse, generating the ~40 kDa cleaved form and high-MW covalent complexes. Lyse directly into boiling 1× Laemmli buffer with DTT, or use ice-cold RIPA supplemented with a broad protease inhibitor cocktail plus 1 mM PMSF and 10 µM E-64. Avoid freeze-thaw of native lysates. For complex analysis (e.g., Serpin-B1:elastase), lyse without reducing agent first, then split and add DTT for parallel reduced/non-reduced blots.

Can I use this antibody for IHC on FFPE tissue, and what antigen retrieval works?

Yes — the antibody is validated for IHC. For FFPE sections, heat-induced epitope retrieval in 10 mM citrate buffer pH 6.0 (20 minutes at sub-boiling) is our standard starting condition; Tris-EDTA pH 9.0 also works and may give cleaner background on lung and gut sections. Start at 1:100-1:200 for IHC-P and titre. Expect strong cytoplasmic staining in neutrophils, monocytes, and mucosal epithelium. Tonsil and bone marrow are reliable control tissues. Include a no-primary control; endogenous peroxidase in granulocytes can give false signal if not quenched adequately.

How should the antibody be stored, and is it stable to repeated freeze-thaw?

Supplied at 1 mg/mL in PBS with 0.02% sodium azide and 50% glycerol. Store at -20°C for long-term; the glycerol formulation means it will not freeze solid, so aliquoting is optional but recommended if you anticipate frequent use over months. For routine bench work, a 4°C working aliquot is stable for at least 4 weeks. Avoid repeated freeze-thaw of the main stock — more than 5 cycles can reduce titre, particularly for IHC applications. Do not store diluted working solutions; prepare fresh from concentrated stock for each experiment.