Anti-Serpin-A12 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Serpin-A12 on Western blot?

Serpin-A12 migrates at approximately 47 kDa on reducing SDS-PAGE, corresponding to the 414-amino acid mature protein. The predicted molecular weight from sequence is around 45 kDa, but glycosylation—Serpin-A12 contains N-glycosylation sites—typically shifts the apparent mass upward by 2-3 kDa. In adipose tissue and serum samples, you may observe a single clean band. If you see higher-molecular-weight species, consider dimers or aggregates; Serpin-A12 can form complexes with its target protease KLK7. Reducing conditions and freshly prepared lysates help minimize artifact bands.

Which tissues or cell lines express Serpin-A12 at detectable levels?

Serpin-A12 was originally identified in visceral adipose tissue and remains most abundant there, particularly in white adipose depots from individuals with obesity or metabolic syndrome. Human primary adipocytes and differentiated 3T3-L1 cells are reliable positive sources. The protein is also expressed in skin, especially in keratinocytes and in lesional psoriatic epidermis. Circulating Serpin-A12 can be detected in serum or plasma. Liver, pancreatic islets, and hypothalamus show lower but measurable expression. Standard HeLa, HEK293, or NIH-3T3 lysates typically yield low or undetectable signal, so adipocyte or skin-derived samples serve as better positive controls.

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

We recommend starting at 1:1000 for Western blot with this rabbit polyclonal. That dilution has been validated against human Serpin-A12 in adipose tissue lysates and works well with standard chemiluminescent detection. If your sample has low Serpin-A12 abundance—for example, non-adipose tissues or serum—you may need to increase antibody concentration to 1:500 or load more total protein. Conversely, overloaded adipocyte lysates can saturate signal at 1:1000; in that case titrate down to 1:2000. Block in five percent non-fat milk or BSA; we have not observed meaningful differences between the two for this target.

Does this antibody cross-react with mouse or rat Serpin-A12?

This antibody is validated for human Serpin-A12. Mouse and rat orthologs share approximately seventy to seventy-five percent sequence identity with human, concentrated in the serpin domain, so cross-reactivity is possible but not guaranteed. We have not formally validated reactivity in rodent samples. If you are working with mouse adipose tissue or 3T3-L1 cells, we suggest running a pilot blot at 1:500 to 1:1000 and including a human positive control in parallel. Alternatively, consider species-matched antibodies if robust rodent data are critical to your experiments. Predicted cross-reactivity based on epitope homology remains speculative without empirical confirmation.

Can I use this Serpin-A12 antibody for immunohistochemistry or immunofluorescence?

Yes. Triple Point Biologics antibodies are routinely validated for immunohistochemistry and immunofluorescence in addition to Western blot. For IHC on paraffin-embedded human adipose or skin sections, antigen retrieval—typically citrate buffer, pH six, with heat—improves signal. Start at 1:100 to 1:200 dilution and optimize from there. For immunofluorescence on fixed cells or frozen sections, 1:50 to 1:100 is a reasonable starting point. Because Serpin-A12 is a secreted protein, you may observe cytoplasmic and extracellular staining. Include a no-primary-antibody control and, if possible, a Serpin-A12 knockout or knockdown sample to confirm specificity.

What are good positive and negative controls for Serpin-A12 experiments?

For a positive control, use lysate or sections from human visceral adipose tissue, differentiated adipocytes, or lesional psoriatic skin; all express Serpin-A12 at easily detectable levels. Recombinant human Serpin-A12 protein also works, though commercial availability varies. Negative controls include non-adipose cell lines with negligible expression—HeLa, HEK293—and omission of primary antibody or substitution with non-immune rabbit IgG at matched concentration. If you have access to SERPINA12-knockdown cells or serum from lean, healthy donors—where circulating Serpin-A12 is lower—these provide biological negatives. Pre-adsorption with recombinant Serpin-A12 peptide, if available, offers an additional specificity check.

How should I store this antibody and how long does it remain stable?

Store the antibody at minus twenty degrees Celsius as supplied. The 100-microgram aliquot is stable for at least two years under these conditions; we date each lot accordingly. For frequent use, prepare small working aliquots—ten to twenty microliters—to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregate formation over time. If you use the antibody weekly, a working stock can be held at four degrees Celsius in the presence of zero-point-zero-two to zero-point-one percent sodium azide for up to one month. Do not store diluted antibody in blocking buffer for more than a few days. Centrifuge briefly before use if you observe any precipitate.

Does Serpin-A12 form complexes with its target protease that affect detection?

Yes. Serpin-A12 irreversibly inhibits kallikrein-related peptidase 7 by forming a covalent serpin-enzyme complex, a hallmark of the serpin mechanism. In samples containing active KLK7—such as skin lysates or co-transfected cell systems—you may detect a higher-molecular-weight band around 75 to 80 kDa corresponding to the Serpin-A12–KLK7 complex in addition to the 47-kDa monomer. This is biologically relevant, not an artifact. Under reducing conditions the covalent complex remains intact. If you want to assess only free Serpin-A12, consider treating samples with a reducing agent and boiling, although the complex is remarkably stable even then.