Serpin A-12 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin A-12 on SDS-PAGE and Western blot?

The SERPINA12-encoded polypeptide is 414 amino acids with a predicted unglycosylated MW of ~47 kDa. Because this recombinant is produced in HEK293 cells under native secretory conditions, N-linked glycosylation shifts the apparent MW on reducing SDS-PAGE to approximately 55–60 kDa. On Western blot using our matched rabbit polyclonal (RP-SerpinA12), you should expect a diffuse band in that 55–60 kDa range, consistent with glycoprotein heterogeneity. Deglycosylation with PNGase F will collapse this to the ~47 kDa core polypeptide if a sharper reference band is needed.

Is recombinant Serpin A-12 (vaspin) produced as a full-length protein or a processed isoform?

This recombinant corresponds to the full-length mature secreted form of Serpin A-12 (UniProt Q8IW75, residues 24–414 after signal peptide cleavage). No reactive-center loop (RCL) pre-cleavage or latency-inducing modifications are introduced during production. The protein is purified in its native, inhibitory-competent conformation and has not been converted to the cleaved or latent serpin form, which is important to confirm before kinetic experiments — cleaved serpin runs ~2–3 kDa smaller on SDS-PAGE under reducing conditions.

What protease does Serpin A-12 (vaspin) inhibit and what substrate can I use to measure KLK7 inhibition activity?

Serpin A-12 inhibits kallikrein-related peptidase 7 (KLK7) via a covalent, irreversible suicide-substrate mechanism. To measure inhibitory activity, pre-incubate Serpin A-12 with recombinant KLK7 for 15–30 min at 37 °C in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, then add the fluorogenic substrate Suc-LLVY-AMC (or Ac-Lys-Pro-Arg-AMC for higher KLK7 selectivity) at 100–200 µM. Monitor fluorescence at Ex 380 nm / Em 460 nm. Residual KLK7 activity relative to a no-inhibitor control directly reports Serpin A-12 inhibitory efficiency.

What starting concentration of recombinant Serpin A-12 should I use for a KLK7 inhibition or IC50 assay?

For inhibition constant (Ki) determination, titrate Serpin A-12 across a 0.1–10× molar excess relative to your KLK7 concentration; a practical starting range is 50–500 nM Serpin A-12 with 50–100 nM KLK7. For competitive IC50 experiments where Serpin A-12 is the reference inhibitor, fix it at your empirically determined Ki and vary the competitor concentration. Because serpins bind stoichiometrically rather than catalytically, mass-based excess rather than catalytic excess determines titration endpoints — keep this in mind when designing dilution series.

Can I use recombinant Serpin A-12 as a positive control for Western blot with the RP-SerpinA12 rabbit polyclonal antibody?

Yes, and this is one of the most straightforward use cases. Load 50–200 ng of this recombinant per lane alongside your cell or tissue lysate. The matched rabbit polyclonal antibody (RP-SerpinA12; /anti-serpin-a12-rabbit-polyclonal-antibody) is guaranteed compatible — both the recombinant and antibody originate from the same TPB program. At 50–100 ng loaded, you should see a clean 55–60 kDa band under standard reducing, denaturing conditions. This also serves as a reliable molecular weight reference for interpreting endogenous vaspin bands in adipose tissue lysates.

How much recombinant Serpin A-12 should I load for Western blot antibody validation with RP-SerpinA12?

For antibody validation purposes — particularly if you are building an antibody validation data set or submitting to a journal requiring positive controls — load a two-point titration: 50 ng and 200 ng per lane. At 50 ng, RP-SerpinA12 should produce a detectable band with signal-to-noise suitable for publication. At 200 ng, band intensity confirms linearity. Running these alongside a KO or knockdown lysate (negative control) satisfies most antibody validation guidelines. The recombinant's purity of >90–95% by SDS-PAGE ensures the band you detect is not a contaminating co-purified protein.

What buffer should I use for diluting or reconstituting recombinant Serpin A-12 for cell-based or biochemical assays?

Recombinant Serpin A-12 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For biochemical inhibition assays, dilute directly into your assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl is fully compatible). For cell-based or adipokine-treatment experiments, buffer-exchange into PBS using a 10 kDa MWCO spin concentrator to remove glycerol before addition to cell culture media. Avoid diluting more than 1:20 into low-ionic-strength buffers without carrier protein (0.1% BSA), as serpins can adsorb to plastic at sub-nanomolar concentrations.

What are the storage and shelf life recommendations for recombinant Serpin A-12 to preserve inhibitory activity?

Store at -20 °C in the single-use aliquots provided. Repeated freeze-thaw cycles degrade serpin inhibitory activity disproportionately relative to total protein, because even partial RCL cleavage or loop insertion during denaturation produces latent or cleaved forms that are antigenically intact but functionally inert. Under recommended storage conditions, inhibitory activity is stable for at least 12 months. If working aliquots are needed, keep at 4 °C for no more than 5–7 days. Confirm functional integrity before key experiments by running a KLK7 inhibition assay alongside a reference aliquot stored from first thaw.