Anti-RECK Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for RECK on a Western blot?

Full-length RECK migrates at approximately 110–125 kDa on reducing SDS-PAGE, significantly above its predicted mass of 107 kDa due to extensive N-glycosylation. The protein contains multiple consensus glycosylation sites that shift its apparent molecular weight. You may observe a diffuse band rather than a single sharp line, reflecting glycan heterogeneity. Treatment with PNGase F or tunicamycin will collapse the band closer to the predicted size. If you see bands near 50–60 kDa, consider proteolytic cleavage during lysis; include protease inhibitors and work quickly on ice.

Does RECK require special sample preparation because it is GPI-anchored?

Yes. RECK is tethered to the plasma membrane via a glycosylphosphatidylinositol anchor, so complete solubilization requires detergent-based lysis buffers. RIPA or buffers containing 1% Triton X-100 or NP-40 work well. Avoid purely aqueous extraction, which will leave RECK in the pellet. For immunofluorescence, standard methanol or paraformaldehyde fixation is sufficient because the epitope remains membrane-associated. If you plan to study secreted or shed RECK fragments, collect conditioned media separately; phospholipase cleavage can release the ectodomain into the supernatant under certain conditions.

What dilution should I start with for Western blot and IHC using this RECK antibody?

For Western blot, begin at 1:1000 in 5% non-fat milk or BSA in TBST, the manufacturer-recommended dilution for this rabbit polyclonal. Overnight incubation at 4°C often improves signal for lower-abundance targets like RECK. For IHC on paraffin sections, a typical starting range is 1:100 to 1:200 after antigen retrieval in citrate buffer, pH 6.0. Because RECK expression varies widely by tissue and tumor type, titrate the antibody against your specific sample. Include a known RECK-positive control, such as normal lung or brain tissue, to benchmark staining intensity.

Is this antibody predicted to cross-react with mouse or rat RECK?

Human RECK shares approximately 85% amino acid identity with mouse and 84% with rat orthologs, particularly high conservation across the Kazal domains and the C-terminal GPI-anchor signal. Cross-reactivity with mouse and rat is therefore predicted but not formally validated by the manufacturer for this lot. If you work with rodent models, run a pilot blot with mouse or rat tissue lysate alongside human controls. Many commercial RECK antibodies do recognize rodent RECK, but band intensity and background may differ. Confirm the expected molecular weight and consider a species-matched positive control from brain or lung.

What positive and negative control cell lines should I use to validate RECK expression?

RECK is broadly expressed but highly variable. For a strong positive control, use non-transformed fibroblasts, normal human brain lysate, or endothelial cells; RECK is critical for CNS angiogenesis and blood-brain barrier maintenance. Many cancer cell lines downregulate RECK during metastatic progression, so aggressive tumor lines such as HT-1080 fibrosarcoma or MDA-MB-231 breast cancer cells typically show low or absent signal and serve as functional negative controls. If you are studying Wnt signaling, compare RECK levels in cells with and without exogenous Wnt7A stimulation to assess co-receptor complex formation.

Can I use this RECK antibody for immunoprecipitation to pull down the Wnt7–frizzled complex?

This polyclonal is validated for Western blot, but immunoprecipitation performance is not specified. Polyclonal antibodies often work for IP if the epitope remains accessible in native conformation. Because RECK forms a ternary complex with ADGRA2 and frizzled receptors, use gentle lysis conditions—avoid SDS and high salt—and include protease and phosphatase inhibitors. Pre-clear lysates with protein A/G beads to reduce background. After IP, you can probe for co-precipitated frizzled-4, ADGRA2, or Wnt7A/B. Run a no-antibody bead-only control to assess nonspecific binding of these interaction partners.

How should I store this RECK antibody and what is the expected shelf life?

Store the antibody at –20°C in single-use aliquots to avoid freeze-thaw cycles, which can reduce titer and increase aggregation. Glycerol or BSA in the formulation buffer helps stabilize rabbit polyclonals during freezing. Once thawed, an aliquot remains stable at 4°C for up to one month; add 0.02% sodium azide if you plan repeated use over several weeks. Do not store diluted working solutions; prepare fresh each time. Under these conditions, the antibody typically retains full activity for at least two years from the manufacturing date. Check for precipitate before use and centrifuge briefly if cloudiness appears.

Why might I see multiple bands for RECK on a Western blot?

Multiple bands usually reflect glycosylation heterogeneity or proteolytic processing. The mature 110–125 kDa species is heavily N-glycosylated, and different cell types add variable glycan structures, creating a ladder or smear. Lower molecular weight bands around 50–80 kDa may represent cleavage products generated by metalloproteinases or other proteases during sample handling, particularly if lysis was slow or samples were not kept cold. RECK can also undergo regulated intramembrane proteolysis. To confirm specificity, compare band patterns in RECK-knockout or siRNA-knockdown lysates. PNGase F treatment simplifies the profile and confirms that upper bands are glycoforms.