Anti-Serpin-A4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Serpin-A4 on Western blot?

Serpin-A4 migrates at approximately 58-68 kDa on reducing SDS-PAGE, higher than the predicted 48 kDa from its 427-amino-acid sequence. This discrepancy arises from extensive N-linked glycosylation; Serpin-A4 is a glycoprotein with multiple glycosylation sites that add roughly 10-20 kDa to its apparent molecular weight. Deglycosylation with PNGase F shifts the band closer to the predicted mass. In plasma or serum samples, you may also detect higher-molecular-weight complexes around 110-120 kDa representing Serpin-A4 covalently bound to tissue kallikrein, a hallmark of serpin-mediated protease inhibition. These SDS-stable complexes confirm functional inhibitor activity.

What starting dilution should I use for Western blot with this Serpin-A4 antibody?

Start at 1:1000 dilution in blocking buffer for Western blot, which is the validated dilution for this rabbit polyclonal. Serpin-A4 is a secreted protein present in plasma at moderate concentrations, so signal intensity will depend heavily on sample type. For plasma or serum, 1:1000 typically yields clean bands without requiring further optimization. For tissue lysates or conditioned media where Serpin-A4 may be less abundant, you may need to titrate down to 1:500 or load more total protein per lane. Incubate overnight at 4°C for best results. Block membranes with five percent non-fat milk or BSA in TBST for one hour before antibody incubation.

Does this antibody detect Serpin-A4 in non-human primate samples?

Cross-reactivity with monkey (non-human primate) samples is predicted based on sequence homology but not yet validated in-house. Human Serpin-A4 shares approximately 90-95 percent identity with cynomolgus and rhesus macaque orthologs, particularly within the epitope region targeted by polyclonal antibodies raised against the full ectodomain. If you are working with primate samples, we recommend running a pilot blot with both human and your species of interest side-by-side to confirm signal specificity. Validated reactivity is currently limited to human samples; we encourage users to share cross-reactivity results for catalog updates.

Can I use this antibody to detect the Serpin-A4-kallikrein complex in plasma?

Yes, this antibody recognizes both free Serpin-A4 and the covalent Serpin-A4-kallikrein complex formed during protease inhibition. On Western blot under reducing conditions, you will see the monomeric Serpin-A4 band near 60-68 kDa and a higher-molecular-weight band around 110-120 kDa corresponding to the equimolar complex. The complex is stable in SDS and heat, a defining property of serpin-protease interactions. If your experimental question involves kallikrein activity, detecting both species provides functional readout. For plasma samples, collect into EDTA or citrate and process quickly; avoid prolonged room-temperature incubation, which can artificially drive complex formation or degradation.

What positive control sample should I use for Serpin-A4 Western blot?

Human plasma or serum is the most physiologically relevant positive control, as Serpin-A4 circulates at detectable levels and is well-characterized in these matrices. Dilute plasma 1:50 to 1:100 in sample buffer before loading to avoid overloading albumin and IgG. Alternatively, liver lysates are suitable; Serpin-A4 is synthesized primarily in hepatocytes. Recombinant human Serpin-A4 protein provides a defined control if you need to verify antibody specificity independent of biological variability. Avoid using HEK293 or CHO lysates unless those cells have been transfected to overexpress Serpin-A4, as endogenous expression in standard cell lines is minimal or absent.

Is Serpin-A4 cleaved or processed in plasma samples, and will the antibody detect fragments?

Serpin-A4 circulates predominantly as an intact 60-68 kDa glycoprotein. The reactive center loop is cleaved when Serpin-A4 inhibits tissue kallikrein, but cleavage occurs within the inhibitor while it remains covalently attached to the protease, forming the stable complex you detect at higher molecular weight. In aged or improperly stored samples, you may occasionally see lower-molecular-weight bands representing degradation products, not physiological processing. This polyclonal antibody is raised against a large portion of the mature protein and should recognize most fragments containing the immunogen region. Fresh or properly frozen samples minimize artifactual degradation and give the cleanest blots.

How should I store this Serpin-A4 antibody, and how long is it stable?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The product ships as a 100 µg pack and remains stable for at least twelve months from the date of receipt when stored frozen and handled properly. Once thawed, an aliquot can be kept at 4°C for up to one month if sodium azide (0.02-0.05 percent final) is added as a preservative. Avoid prolonged exposure to room temperature. Do not refreeze thawed aliquots more than twice. If you notice precipitate after thawing, centrifuge briefly at 10,000 x g before use to pellet aggregates that can cause high background.

Will this antibody work for immunohistochemistry on human tissue sections?

Triple Point Biologics antibodies are validated for Western blot applications across the catalog, and this Serpin-A4 antibody is suitable for immunohistochemistry on formalin-fixed paraffin-embedded human tissues. Serpin-A4 expression is prominent in liver hepatocytes and detectable in kidney, vascular endothelium, and other sites where the kallikrein-kinin system is active. For IHC, start with antigen retrieval using citrate buffer pH 6.0 in a pressure cooker or steamer, then dilute the antibody 1:100 to 1:200 for initial trials. Optimize blocking conditions to minimize background. Appropriate positive-control tissues include liver and kidney; negative controls should omit primary antibody or use an isotype control.