Anti-Serpin-B10 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Serpin-B10 on Western blot?

Serpin-B10 migrates at approximately 45 kDa on SDS-PAGE, corresponding to the 397-amino-acid full-length protein. Because Serpin-B10 lacks a signal peptide and is not post-translationally cleaved in the classical serpin manner, you should see a single predominant band in most cell lysates. Nuclear-enriched fractions typically yield stronger signal than whole-cell lysates due to its predominant nuclear localization. If you observe higher-molecular-weight species, consider the possibility of ubiquitinated forms or non-specific binding; running a positive control lysate from a hematopoietic cell line helps distinguish genuine signal from artifact.

What is the recommended starting dilution for Western blot with this Serpin-B10 antibody?

Start at 1:1000 dilution in blocking buffer for Western blot, based on our validation with human cell lysates. This rabbit polyclonal typically produces clean signal at that dilution when loading 20–30 µg total protein per lane. If your expression system or cell type shows low endogenous Serpin-B10, you may improve sensitivity by concentrating to 1:500. Conversely, if background is high, dilute further to 1:2000. Always include a no-primary-antibody control lane to assess non-specific binding of your secondary reagent, particularly important with polyclonal antibodies.

Is this antibody validated for immunohistochemistry and immunofluorescence on Serpin-B10?

Yes. Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry as standard practice. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required; citrate buffer at pH 6.0 is a reasonable starting point for nuclear antigens like Serpin-B10. For immunofluorescence, expect nuclear and perinuclear staining patterns consistent with Serpin-B10 localization. We recommend empirical titration starting at 1:100 to 1:200 for both IHC and IF, as optimal dilution depends on fixation time, permeabilization, and detection chemistry. Methanol fixation often preserves nuclear epitopes better than paraformaldehyde for IF.

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

Clade B serpins share structural homology, so cross-reactivity with closely related family members such as Serpin-B9 or Serpin-B6 is a valid concern. This polyclonal was raised against a region spanning residues in the Serpin-B10 sequence, but we have not systematically tested all 13 human clade B serpins for cross-reactivity. If your experimental system co-expresses multiple Serpin-B isoforms, run lysates from cells with known single-serpin expression or use siRNA knockdown as a specificity control. The predicted molecular weights of other clade B serpins range from 42 to 55 kDa, so band size alone is insufficient to confirm identity.

Will this Serpin-B10 antibody work in mouse or rat samples?

This antibody is validated in human samples and predicted to cross-react broadly across mammals, with dog listed as a potential cross-reactive species. Mouse and rat Serpin-B10 orthologs share approximately 70–75 percent sequence identity with human, so cross-reactivity is plausible but not guaranteed. If you are working with rodent models, we recommend testing the antibody at 1:500 and 1:1000 dilutions on a small-scale pilot blot with positive-control lysate from a tissue known to express Serpin-B10, such as bone marrow or spleen. Include a blocking peptide or knockout lysate if available to confirm specificity.

What sample preparation tips improve Serpin-B10 detection in nuclear fractions?

Because Serpin-B10 is predominantly nuclear, subcellular fractionation improves signal-to-noise substantially. Use a gentle lysis buffer with low detergent concentration for the cytoplasmic fraction, then extract nuclei with a higher-salt buffer containing 300–400 mM NaCl to solubilize nuclear proteins. Include protease inhibitors throughout; serpins themselves are protease inhibitors but can still be substrates for certain proteases during lysis. Avoid over-sonicating nuclear pellets, which can shear chromatin and complicate downstream analysis. Verify fractionation quality by blotting for Lamin B1 or histone H3 as nuclear markers and tubulin or GAPDH for cytoplasmic markers.

How should I store this Serpin-B10 antibody to maintain long-term stability?

Store the antibody at –20 °C as a glycerol stock or in small single-use aliquots to avoid repeated freeze-thaw cycles, which can aggregate immunoglobulins and reduce titer. If your workflow requires frequent access, a working aliquot can be kept at 4 °C for up to one month in the presence of 0.02–0.05 percent sodium azide as a preservative. Do not store diluted antibody in blocking buffer for more than a few days; the carrier proteins in milk or BSA-based blockers can promote microbial growth. For long-term archive storage beyond one year, –80 °C is preferable, particularly for polyclonal sera.

What positive control cell line or tissue expresses high levels of Serpin-B10?

Serpin-B10 is expressed in hematopoietic tissues and certain myeloid cell lines. Bone marrow, spleen, and thymus are reliable tissue sources for positive controls in IHC. For Western blot, consider using lysates from U937, THP-1, or HL-60 myeloid cell lines, which express detectable Serpin-B10. Some epithelial cells and fibroblasts express lower levels, so HeLa or HEK293 lysates may serve as weak-positive or negative controls. If you are studying TNF-induced apoptosis pathways, pre-treating cells with TNF-alpha for 4–6 hours can modulate Serpin-B10 levels and provide an inducible control for antibody specificity.