Anti-Leishmanolysin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for LMLN/invadolysin on a Western blot?

Full-length human LMLN is 647 amino acids with a predicted molecular weight of approximately 73 kDa. On Western blot of human lysates, the dominant band typically migrates between 70 and 75 kDa under reducing conditions. Depending on cell type and processing state, you may also observe a lower band around 55-60 kDa corresponding to the mature, propeptide-cleaved form, since LMLN contains an N-terminal propeptide that regulates enzymatic activity. Glycosylation status and sample handling can shift apparent mobility by a few kDa. Run a positive control lysate (HeLa or HEK293 are reasonable starting points) alongside your samples.

Why am I seeing multiple bands when blotting for LMLN?

LMLN is processed from a zymogen: the propeptide domain is removed to yield the active metalloprotease, so a doublet near 73 kDa (pro-form) and 55-60 kDa (mature) is biologically plausible rather than nonspecific signal. Additional bands can reflect alternative splice variants, post-translational modification, or partial proteolysis during lysis. To distinguish specific from nonspecific signal, run an siRNA or CRISPR LMLN-knockdown control, include a no-primary control, and ensure samples are kept cold with protease inhibitors (avoid EDTA if you also want to preserve zinc-dependent activity for downstream assays).

What dilution should I start with for Western blot?

Start at 1:1000 in 5% non-fat milk or BSA in TBST, with overnight incubation at 4°C. This is the validated starting point for this rabbit polyclonal on human lysates. If background is high, titre down to 1:2000 or 1:5000; if signal is weak in low-expressing lines, go to 1:500 and extend exposure rather than over-loading lysate. LMLN expression varies considerably across cell lines, so empirical titration against your specific lysate is recommended. Load 20-40 µg total protein per lane as a starting range.

Does this antibody work for IHC and immunofluorescence as well as Western blot?

The antibody is validated brand-wide for Western blot, though the dilution provided (1:1000) is specific to WB. For IHC on FFPE sections, start with heat-induced epitope retrieval in citrate buffer (pH 6.0) and titre the primary in the 1:100-1:500 range. For IF, 1:100-1:200 on methanol- or paraformaldehyde-fixed cells is a reasonable starting window. LMLN is reported as cytoplasmic, so expect diffuse cytosolic staining; some studies also describe association with chromosomes during mitosis, which can serve as an internal localization check.

What species does this antibody react with?

Reactivity is validated against human LMLN. Cross-reactivity with mouse and rat orthologs is predicted based on sequence homology in the immunogen region but has not been confirmed in-house. Human LMLN shares roughly 90% identity with mouse Lmln across the catalytic domain, so detection in rodent samples is plausible but should be confirmed with a knockdown or knockout control before being used quantitatively. If you need validated rodent reactivity, request lot-specific data or run a pilot blot on mouse tissue alongside a human positive control.

What positive and negative controls should I include?

For positive controls, HeLa, HEK293, and several cancer lines (including hepatocellular and breast carcinoma lines) express detectable LMLN by Western blot. For a negative or loss-of-function control, siRNA knockdown of LMLN in your cell line of interest is the most rigorous option; published siRNA sequences targeting LMLN exon 2 work well. Recombinant human LMLN protein can serve as a size marker if available. Because LMLN has been linked to drug resistance phenotypes, consider running paired parental and resistant lines if that is relevant to your project.

How should I store the antibody and how stable is it?

Store the 100 µg aliquot at -20°C for long-term storage. Upon first thaw, aliquot into single-use volumes (5-10 µL) to avoid repeated freeze-thaw cycles, which degrade polyclonal performance over time. Working aliquots can be kept at 4°C for up to two weeks in the presence of the supplied preservative. Avoid storage in frost-free freezers where temperature cycling occurs. The antibody is supplied in a standard PBS-based buffer with glycerol and sodium azide; note that azide inhibits HRP, so wash thoroughly before adding HRP-conjugated secondaries.

Is the immunogen in the propeptide or catalytic domain, and does that affect what I detect?

This rabbit polyclonal was raised against a recombinant fragment spanning the central region of human LMLN, which includes sequence from both the propeptide and the catalytic metalloprotease domain. In practice, this means the antibody recognizes both the unprocessed zymogen (~73 kDa) and the mature, propeptide-cleaved form (~55-60 kDa), which is useful if you are tracking activation status. If you need to distinguish the two forms unambiguously, pair this antibody with a propeptide-specific reagent or use mobility on a 10% gel with extended run time to resolve the doublet.