Anti-Serpin-A10 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-A10 on Western blot?

Serpin-A10 migrates at approximately 52-55 kDa on reducing SDS-PAGE, slightly higher than the predicted mass of 49 kDa for the 444-amino acid mature protein. This discrepancy reflects glycosylation; the protein contains N-linked glycosylation sites that contribute to its apparent molecular weight. You may observe a single distinct band in plasma or liver lysates, where expression is highest. If you see multiple bands, consider whether your sample contains proteolytic fragments or post-translational variants. We recommend starting at 1:1000 dilution for Western blot with standard ECL detection on 20-30 micrograms of lysate per lane.

Is this Serpin-A10 antibody specific for the protein Z-dependent form or does it detect free Serpin-A10?

This polyclonal antibody recognizes Serpin-A10 regardless of cofactor binding status. It detects both free Serpin-A10 and the protein Z-bound complex, since the epitopes span the full-length protein rather than the cofactor interaction interface. In plasma samples, most Serpin-A10 circulates in free form under physiological conditions; complex formation occurs transiently during coagulation activation. For functional studies distinguishing bound versus free forms, you will need immunoprecipitation followed by co-detection of protein Z, or surface plasmon resonance approaches. The antibody itself does not differentiate these states by Western blot or immunohistochemistry.

Does this antibody cross-react with other serpins like antithrombin or protein C inhibitor?

Cross-reactivity with other serpin family members has not been systematically validated. Serpin-A10 shares structural homology with other clade A serpins, particularly in the reactive center loop region, but sequence identity is typically below 40 percent with antithrombin or protein C inhibitor. Given that this is a rabbit polyclonal raised against a broad immunogen, some cross-reactivity is theoretically possible in crude lysates containing high concentrations of abundant serpins. We recommend running recombinant Serpin-A10 alongside your experimental samples and including a negative control lysate from a Serpin-A10-low tissue to confirm band specificity during initial validation.

What sample types and preparation methods work best for detecting Serpin-A10?

Serpin-A10 is a secreted plasma protein, so it is readily detected in serum, citrated plasma, or conditioned media from hepatocyte cultures. For tissue lysates, liver shows the highest expression, followed by kidney. Use standard RIPA or NP-40 lysis buffers with protease inhibitors; avoid prolonged incubation at room temperature, as Serpin-A10 can be cleaved by endogenous proteases. For immunohistochemistry, citrate-based antigen retrieval at pH 6.0 typically yields optimal staining in formalin-fixed paraffin-embedded liver sections. Frozen sections work well without retrieval. Serpin-A10 plasma concentration is relatively low compared to antithrombin, so load adequate protein.

What dilution should I start with for IHC on human liver tissue?

Begin with a 1:200 to 1:500 dilution for immunohistochemistry on formalin-fixed paraffin-embedded human liver sections following heat-mediated antigen retrieval in citrate buffer. Serpin-A10 expression is primarily hepatocellular, with strongest signal in periportal hepatocytes. Overnight incubation at 4 degrees Celsius often improves signal-to-noise ratio compared to shorter room-temperature incubations. Titre the antibody across a dilution series on your specific tissue batch, as fixation time and antigen retrieval conditions influence optimal working concentration. A negative control using rabbit IgG at matched concentration is essential, given that liver contains endogenous immunoglobulin-binding proteins.

Can I use this antibody to detect mouse or rat Serpin-A10?

This antibody is validated for human Serpin-A10. Cross-reactivity with mouse or rat orthologs is predicted but not experimentally confirmed. Human and mouse Serpin-A10 share approximately 70 percent amino acid identity, with highest conservation in the core serpin domain. If you are working with rodent samples, we recommend testing the antibody at the standard 1:1000 dilution alongside a positive human control. Some researchers report detectable signal in mouse plasma by Western blot, but band intensity may be reduced and specificity should be confirmed with a knockout or knockdown control. For definitive rodent studies, consider a species-specific antibody.

How should I store the antibody and what is the expected shelf life?

Store the antibody at minus 20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. The antibody is supplied in a glycerol-containing buffer that remains liquid at minus 20 degrees; vortex gently after thawing to ensure homogeneity. For short-term use within two weeks, the working dilution can be kept at 4 degrees Celsius with 0.02 percent sodium azide as preservative. Under these conditions, expect stable performance for at least 12 months from manufacture date. Avoid prolonged exposure to room temperature and do not store diluted antibody without azide for more than a few days.

What positive and negative controls should I include when using this Serpin-A10 antibody?

For Western blot, human plasma or serum serves as an excellent positive control, as does lysate from HepG2 hepatoma cells, which express Serpin-A10. Recombinant Serpin-A10 provides a definitive molecular weight reference. As a negative control, use lysate from a tissue with minimal expression, such as skeletal muscle or brain, or employ siRNA knockdown in a hepatocyte line. For immunohistochemistry, normal human liver is the gold-standard positive control; include a serial section incubated with rabbit IgG isotype control at equivalent concentration to assess nonspecific binding. These controls confirm antibody specificity and help troubleshoot unexpected results.