Hemicentin (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Hemicentin-1 run at on SDS-PAGE and Western blot?

Full-length human Hemicentin-1 (HMCN1, UniProt Q96RW7) has a predicted molecular weight of approximately 614 kDa from its 5,635 amino acid sequence. On SDS-PAGE under reducing conditions, the recombinant produced in HEK293 cells typically migrates anomalously above 460 kDa due to extensive N- and O-linked glycosylation inherent to mammalian-expressed ECM proteins. Do not expect a sharp, tight band at the sequence-predicted MW — diffuse, smeared signal in the 460–700 kDa range is normal and reflects native-like glycan decoration rather than degradation or aggregation.

Is this recombinant full-length Hemicentin-1 or a fragment, and which isoform does it correspond to?

This recombinant corresponds to the canonical full-length isoform 1 of human Hemicentin-1 (UniProt Q96RW7-1, 5,635 aa), expressed in HEK293 cells with a signal peptide removed post-translationally to yield the secreted mature form. No further proteolytic processing sites are definitively mapped in the literature for human Hemicentin-1 under physiological conditions. The product is supplied as the secreted, glycosylated ectodomain in solution. Researchers working with shorter constructs or specific domain fragments (e.g., isolated Ig-like repeats) should note this preparation covers the full extracellular span.

What is the domain architecture of Hemicentin-1 and does it bind or cleave any substrates?

Hemicentin-1 is not a protease and does not cleave substrates. Its domain architecture includes an N-terminal fibulin-type module, 48 tandem immunoglobulin-like repeats, multiple EGF-like domains, and a C-terminal fibulin module — collectively mediating protein–protein and protein–matrix interactions rather than catalytic activity. Functionally, it organises basement membrane architecture and stabilises cell–cell junctions. In binding assays, it interacts with other ECM components such as fibronectin and perlecan. Use this recombinant as a coating substrate, soluble ligand, or interaction partner in pull-down and ELISA-based binding experiments rather than enzymatic activity assays.

What concentration should I use to coat plates or use as a substrate for cell adhesion assays with recombinant Hemicentin-1?

For cell adhesion and spreading assays, a starting coating concentration of 2–10 µg/mL in PBS (applied overnight at 4°C, then blocked with 1% BSA) is a reasonable initial range. Given Hemicentin-1's role as a large ECM scaffold protein, concentrations at the lower end (2–5 µg/mL) often saturate binding sites on standard tissue-culture plastic; titrate per cell type. For pull-down or co-immunoprecipitation experiments using this protein as bait, 1–5 µg per reaction in 50 mM Tris-HCl pH 7.5, 150 mM NaCl is a practical starting point consistent with the supplied storage buffer.

What buffer is recombinant Hemicentin-1 supplied in and how should I dilute it for experiments?

Recombinant Hemicentin-1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This buffer is compatible with most cell-based and biochemical assays without further exchange. For cell-based work, dilute directly into serum-free culture medium; the glycerol concentration drops to negligible levels at working dilutions (≥1:10). If your assay is sensitive to glycerol (e.g., SPR/BLI binding kinetics), buffer-exchange via centrifugal ultrafiltration (100 kDa MWCO) into assay-appropriate buffer is recommended. Avoid PBS alone for long-term diluted stock storage — the Tris-NaCl-glycerol formulation maintains protein stability at -20°C.

How should I store recombinant Hemicentin-1 and what is its shelf life after thawing?

Store at -20°C in single-use aliquots as supplied. Avoid repeated freeze-thaw cycles, which risk aggregation of this large glycoprotein (>460 kDa on gel). Once thawed, keep on ice and use within 24 hours; do not refreeze. Aliquots stored continuously at -20°C are stable for at least 12 months from the date of receipt when kept in the original formulation (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Adding a carrier protein such as 0.1% BSA is not necessary given the glycerol-containing buffer, but may help at very low working concentrations (<0.1 µg/mL).

Can I use recombinant Hemicentin-1 as a Western blot positive control for the RP-Hemicentin rabbit polyclonal antibody?

Yes — this is a validated pairing. Recombinant Hemicentin-1 (REC-Hemicentin) was used during the development and lot-release testing of the matched RP-Hemicentin rabbit polyclonal antibody, confirming band detection by Western blot. Load 50–200 ng of recombinant per lane on a 3–8% Tris-acetate or gradient gel (standard SDS-PAGE resolving gels are inadequate for a protein migrating above 460 kDa). Expect a diffuse band in the 460–700 kDa region under reducing conditions. This lane serves as a clean, cell-lysate-free positive control for antibody validation experiments and troubleshooting signal in tissue lysates.

How much recombinant Hemicentin-1 should I load for a Western blot positive control, and what gel percentage do you recommend?

Load 100–200 ng per lane as an initial titration; signal intensity can be adjusted to 50 ng if background is high. Because Hemicentin-1 migrates well above 250 kDa, use a 3–8% Tris-acetate polyacrylamide gel or a 4–12% gradient gel run at low voltage to allow adequate separation in the high-MW range. Pair with the RP-Hemicentin antibody (SKU: RP-Hemicentin) at the manufacturer's recommended dilution. Transfer efficiency for proteins of this size is the most common bottleneck — use a wet-tank transfer system with 0.025% SDS added to transfer buffer, and a PVDF membrane (0.45 µm pore size).