Anti-Myocilin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Myocilin is synthesized as a 55 kDa precursor glycoprotein. Depending on your sample type and processing conditions, you may observe the full-length 55 kDa band, a 35 kDa C-terminal fragment, and a 20 kDa N-terminal fragment resulting from proteolytic cleavage. Trabecular meshwork cells and aqueous humor often show both precursor and processed forms. Glycosylation can shift apparent molecular weight slightly upward. If you see multiple bands, consider whether your tissue expresses active proteases that cleave myocilin. Running a reducing gel with β-mercaptoethanol is recommended, as myocilin contains disulfide bonds in the olfactomedin domain.

What starting dilution should I use for myocilin Western blot with this antibody?

We recommend starting at 1:1000 dilution for Western blot based on validation with human trabecular meshwork lysates. Overnight incubation at 4°C typically yields clean signal with low background. If your lysate has low myocilin expression or you are working with conditioned media where myocilin is secreted, you may need to concentrate samples or increase antibody concentration to 1:500. Always include a positive control such as trabecular meshwork or ciliary body lysate. Titrate against your specific sample type, as myocilin levels vary widely across tissues and can be induced by dexamethasone treatment in responsive cell lines.

Does this myocilin antibody cross-react with mouse or rat samples?

This antibody is validated for human myocilin. Cross-reactivity with mouse or rat is not experimentally confirmed. Human myocilin shares approximately 87% amino acid identity with mouse and 85% with rat orthologs, suggesting potential cross-reactivity, but epitope accessibility can differ. If working with rodent samples, we recommend testing the antibody on a known positive control lysate from mouse or rat trabecular meshwork or retina alongside human lysate. Alternatively, consider a species-specific antibody if consistent detection in non-human models is critical to your experimental design. Predicted cross-reactivity should always be empirically validated in your system.

Which tissues or cell lines are good positive controls for myocilin expression?

Trabecular meshwork cells are the gold standard positive control, as myocilin was originally identified as trabecular meshwork-induced glucocorticoid response protein. Primary human trabecular meshwork cells or trabecular meshwork tissue lysates express constitutive myocilin that increases robustly upon dexamethasone treatment. Other useful controls include ciliary body, sclera, and optic nerve head tissues. For cell lines, consider using trabecular meshwork cell strains treated with 100–1000 nM dexamethasone for 3–7 days to induce myocilin expression. Bone and skeletal muscle also express myocilin at lower levels. Negative controls can include HeLa or HEK293 cells, which typically show minimal endogenous expression.

Can I use this antibody for immunofluorescence or immunohistochemistry on myocilin?

Triple Point Biologics antibodies are validated for Western blot applications as part of our standard workflow. This rabbit polyclonal should be suitable for immunohistochemistry on formalin-fixed paraffin-embedded sections and frozen sections. For IHC, antigen retrieval using citrate buffer pH 6.0 with heat is commonly required for myocilin detection. Start with 1:100 to 1:200 dilution for IHC and immunofluorescence. Myocilin localizes to the cytoplasm and is also secreted into extracellular matrix, so expect both intracellular and extracellular staining depending on tissue type. Always run no-primary-antibody controls to assess non-specific binding of secondary reagents.

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

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg format should be aliquoted upon receipt into working volumes based on your expected usage. Rabbit polyclonal antibodies stored properly at –20°C typically maintain activity for at least two years from the date of manufacture. For short-term use over several weeks, a working aliquot can be kept at 4°C with 0.02% sodium azide as preservative. Avoid storing in frost-free freezers where temperature cycling occurs. If you observe increased background or reduced signal, prepare a fresh aliquot rather than continuing with a degraded stock.

Does myocilin have alternatively spliced isoforms I should be aware of?

The canonical myocilin isoform is 504 amino acids encoded by three exons. Alternative splicing is not a prominent feature of the MYOC gene under normal conditions, and most studies focus on the full-length protein. However, pathogenic mutations in myocilin, particularly in the olfactomedin domain encoded by exon 3, are linked to primary open-angle glaucoma and result in misfolded protein that accumulates intracellularly rather than being secreted. If studying disease-associated variants, Western blot may reveal retention of higher molecular weight aggregates or reduced secretion into media. This antibody, raised against a broad region, should recognize both wild-type and most mutant forms, but aggregation may affect epitope accessibility.

What sample preparation considerations are important for detecting secreted myocilin?

Myocilin is a secreted glycoprotein, so it can be detected in both cell lysates and conditioned culture media or aqueous humor samples. For conditioned media, concentrate samples 10- to 20-fold using centrifugal filters with a 10 kDa molecular weight cutoff to increase signal. Remove serum from culture media during conditioning, as serum proteins interfere with detection. For extracellular matrix preparations, sequential extraction with detergent-containing buffers may be needed. Trabecular meshwork cells secrete myocilin into the extracellular matrix where it associates with fibrillar structures. Use protease inhibitor cocktails during sample preparation to prevent proteolytic cleavage. Compare intracellular and extracellular fractions to assess secretion efficiency, particularly when studying glaucoma-associated mutants that show impaired trafficking.