Anti-Serpin-A3 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-A3 on a Western blot?

Serpin-A3 (alpha-1-antichymotrypsin) typically migrates at approximately 55–68 kDa on reducing SDS-PAGE, though the mature secreted protein comprises 423 amino acids with a calculated mass near 47 kDa. The discrepancy reflects extensive N-glycosylation at multiple sites. You may observe a broad or smeared band due to glycosylation heterogeneity, particularly in plasma or serum samples. Liver lysates and conditioned media from hepatocyte cultures are reliable positive controls. Start with the recommended 1:1000 dilution in 5-percent non-fat milk or BSA; overnight incubation at 4°C often improves sensitivity for this secreted target.

Is this Serpin-A3 antibody validated for human samples only or does it detect mouse and rat?

This polyclonal antibody is validated for human Serpin-A3 detection by Western blot and immunohistochemistry. Cross-reactivity with non-human primate samples is predicted based on sequence homology but remains experimentally unconfirmed in our hands. Mouse and rat orthologues share lower sequence identity in key epitope regions, so cross-reactivity is not expected. If you work with rodent models, consider species-specific reagents or perform a pilot blot with mouse liver lysate alongside human control to empirically test cross-reactivity before committing to a full experiment. We cannot guarantee performance outside validated human applications.

What is the best positive control tissue or cell line for Serpin-A3 Western blots?

Human liver lysate serves as the strongest positive control because hepatocytes are the primary site of Serpin-A3 synthesis under basal and acute-phase conditions. Human plasma or serum diluted in sample buffer also works well, given circulating concentrations of 0.3–0.6 mg/mL at baseline. For cell lines, HepG2 hepatoma lysates typically express detectable Serpin-A3. If studying inflammation or extrahepatic expression, lysates from stimulated monocytes or brain tissue may show inducible signal, though at lower abundance. Always include a no-primary-antibody or isotype control lane to confirm specificity, especially when working with complex tissue extracts.

Can this antibody distinguish between Serpin-A3 isoforms or splice variants?

Human SERPINA3 does not exhibit well-characterized splice variants that produce distinct protein isoforms in most tissues. The primary transcript encodes a single mature 423-amino-acid secreted protein. However, extensive post-translational glycosylation generates microheterogeneity visible as band broadening on Western blots. This polyclonal antibody was raised against a large fragment spanning residues encompassing much of the mature protein, so it will not discriminate between glycoforms. If your experimental question centers on glycosylation state, consider enzymatic deglycosylation with PNGase F prior to SDS-PAGE to collapse the signal into a single sharper band near 47 kDa.

What dilution should I start with for immunohistochemistry on formalin-fixed paraffin-embedded tissue?

We recommend starting at 1:200 to 1:500 for IHC on formalin-fixed paraffin-embedded human tissue sections. Antigen retrieval is typically required; citrate buffer (pH 6.0) heat-mediated retrieval for 20 minutes works well for many serpin targets. Serpin-A3 expression localizes predominantly to hepatocytes in liver and to reactive astrocytes or sites of inflammation in brain. Include a known positive tissue (normal human liver) and a no-primary-antibody control on adjacent sections. Because Serpin-A3 is a secreted protein, expect cytoplasmic and extracellular staining. Optimize blocking and detection reagents to minimize background in connective tissue.

How should I prepare cell lysates to preserve Serpin-A3 for Western blot detection?

Serpin-A3 is a secreted protein, so you may recover more target from conditioned culture medium than from intracellular lysates, particularly in hepatocyte or HepG2 cultures. For lysates, use RIPA or similar detergent buffer supplemented with protease inhibitors; serpin structure is relatively stable, but include a cocktail to prevent artifactual cleavage. Clarify lysates by centrifugation at 16,000 × g for ten minutes at 4°C. Quantify protein by BCA or Bradford and load 20–40 µg total protein per lane. Because Serpin-A3 is glycosylated, avoid prolonged boiling; heat samples at 95°C for five minutes in reducing sample buffer to preserve band sharpness.

Does Serpin-A3 expression increase under inflammatory or acute-phase conditions, and will this antibody detect it?

Yes, Serpin-A3 is a canonical acute-phase reactant; hepatic synthesis increases several-fold in response to interleukin-6 and other inflammatory cytokines. Plasma concentrations can rise from baseline 0.3–0.6 mg/mL to well over 1 mg/mL during infection, trauma, or chronic inflammation. This polyclonal antibody detects both basal and induced Serpin-A3 with equal affinity, so it is well suited for studies comparing resting versus stimulated conditions. When quantifying induction by Western blot, ensure your linear range is not exceeded; you may need to load less total protein or dilute samples from acute-phase plasma to avoid saturating the signal.

How should I store this antibody, and how long will it remain stable after opening?

Store the antibody at –20°C as supplied. After opening, we recommend aliquoting into single-use volumes to avoid repeated freeze-thaw cycles, which can reduce titre over time. Polyclonal antibodies typically retain activity for at least 12 months when stored as frozen aliquots at –20°C. For frequent use, a working aliquot may be kept at 4°C for up to one month in the presence of 0.02-percent sodium azide or similar preservative; verify that azide does not interfere with HRP-based detection in your system. Do not store diluted antibody in blocking buffer for more than a few days. Always centrifuge thawed aliquots briefly before use to pellet any aggregates.