Serpin A-6 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Serpin A-6 on SDS-PAGE and Western blot?

The mature human Serpin A-6 (CBG/Transcortin) protein spans 405 amino acids, giving a predicted unglycosylated MW of ~44 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds substantial mass — expect a diffuse band running at approximately 52–58 kDa on reducing SDS-PAGE, consistent with the native plasma-derived protein. The smearing is characteristic of heterogeneous glycosylation and is not an indicator of degradation. Bacterially expressed or deglycosylated preparations will collapse toward ~44 kDa.

Does recombinant Serpin A-6 include the signal peptide, or is it the mature secreted form?

This preparation represents the mature secreted form of Serpin A-6 (UniProt P08185), with the 22-residue N-terminal signal peptide removed post-translationally during HEK293 secretory processing. The expressed sequence covers residues 23–427 (inclusive of the pro-region), matching the predominant circulating isoform. No additional propeptide cleavage tags have been introduced, so the N-terminus is authentic to native CBG. If your assay requires a specific terminal epitope, confirm antibody epitope coverage against the mature sequence before use.

Does neutrophil elastase cleave Serpin A-6 / CBG and release cortisol — can I replicate that with this recombinant?

Yes. Serpin A-6 contains a reactive center loop (RCL) that is cleaved by neutrophil elastase (HNE) between Thr345 and Leu346, inducing a conformational change that reduces steroid-binding affinity roughly 10-fold. To replicate this in vitro, incubate Serpin A-6 with HNE at a molar ratio of 1:50–1:200 (HNE:CBG) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl at 37°C for 30–60 minutes. RCL-cleaved CBG runs at ~40 kDa on non-reducing SDS-PAGE due to the conformational shift. Cortisol displacement can be confirmed by competitive binding or equilibrium dialysis.

What buffer conditions are recommended for Serpin A-6 steroid-binding affinity assays?

Serpin A-6 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, which is compatible with most equilibrium dialysis and fluorescence displacement assays without buffer exchange. For competitive binding experiments using tritiated cortisol or dexamethasone, dilute into PBS pH 7.4 containing 0.1% BSA to minimize non-specific adsorption to assay plates. Avoid phosphate concentrations >50 mM, which can slightly reduce binding affinity at physiological temperature (37°C). Reported Kd for cortisol binding to native human CBG is ~30–35 nM at 37°C; expect comparable values with this HEK293-derived preparation.

What starting concentration should I use for Serpin A-6 in a cortisol competition binding assay?

For a standard competitive binding assay using radiolabeled cortisol, a Serpin A-6 concentration of 50–200 nM (approximately 2.5–10 µg/mL based on ~52 kDa glycosylated MW) is a practical starting range. This places you near the Kd (~30–35 nM) so competition curves have adequate dynamic range. Verify active protein concentration by a steroid-binding assay rather than relying solely on total protein measurement by BCA, since glycosylation contributes ~20–25% of measured mass but not to binding stoichiometry. Titrate in at least 8 points across a 3-log concentration range for IC50 determination.

Can I use recombinant Serpin A-6 as a Western blot positive control with the matched TPB antibody RP-SerpinA6?

Yes — this recombinant is the intended positive control for the matched rabbit polyclonal antibody RP-SerpinA6 (/anti-serpin-a6-rabbit-polyclonal-antibody), which was raised and validated in the same workflow. Load 20–50 ng of recombinant Serpin A-6 per lane on a 10% SDS-PAGE gel under reducing conditions; expect a primary band at 52–58 kDa. At RP-SerpinA6 dilutions of 1:1,000–1:3,000, signal is typically clean with low background. This pairing is particularly useful when validating antibody lot-to-lot consistency or confirming positive signal in novel cell/tissue lysates where endogenous CBG expression is uncertain.

How much recombinant Serpin A-6 should I load as a positive control lane alongside tissue or plasma lysates?

20–50 ng per lane is sufficient to produce a clearly detectable band with RP-SerpinA6 at 1:1,000–1:2,000 dilution, and this load falls well below the linear saturation range of most HRP-based detection systems. For plasma samples, endogenous CBG runs at 55–60 kDa due to plasma-specific glycoforms, so the recombinant band at 52–58 kDa will co-migrate closely but can be distinguished by lane position. If running hepatocyte or liver lysates where CBG is synthesized, the recombinant control confirms antibody sensitivity against the mature secreted form rather than intracellular intermediates.

How should I store and handle recombinant Serpin A-6 to preserve steroid-binding activity over time?

Store at -20°C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Repeated freeze-thaw cycles progressively denature the RCL and reduce cortisol-binding capacity; even two additional cycles can measurably reduce Kd fidelity. Upon first thaw, centrifuge briefly at 10,000 × g to pellet any micro-aggregates before use. Working dilutions in PBS/0.1% BSA are stable at 4°C for up to 48 hours. Do not dilute into detergent-containing buffers (e.g., 0.1% Tween-20) if steroid-binding activity is the endpoint, as mild detergents can partially unfold the binding pocket.