Anti-Ovastacin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Ovastacin (ASTL) has a predicted molecular weight of approximately 48 kDa based on its 431-residue sequence. However, the observed band may migrate higher due to N-glycosylation, which is common for secreted metalloproteases. In oocyte lysates you may detect the pro-enzyme form, while post-fertilization samples could show processed mature enzyme or cleavage fragments if activation has occurred. Run a positive control lysate from human oocyte-containing tissue alongside your samples, and consider using a reducing gel to resolve disulfide-linked complexes if you see unexpected high-molecular-weight species.

Is this ovastacin antibody validated for immunofluorescence on oocytes?

Triple Point Biologics antibodies are routinely validated for Western blot and immunohistochemistry; immunofluorescence performance depends on epitope accessibility after fixation. For ovastacin IF, start with methanol fixation at minus 20 degrees Celsius or brief paraformaldehyde followed by permeabilization, since ovastacin localizes to cortical granules in the oocyte cytoplasm. Use a starting dilution around 1 to 500 and titrate. Include unfertilized versus fertilized oocytes as biological controls: ovastacin redistributes to the zona pellucida after cortical granule exocytosis, providing an internal spatial control for specificity.

Will this rabbit polyclonal ovastacin antibody work in mouse oocyte samples?

Mouse ovastacin shares substantial sequence identity with human ASTL, so cross-reactivity is predicted but not yet formally validated in our hands. The catalytic astacin domain is highly conserved across mammals. For mouse samples, start with the recommended 1 to 1000 dilution for Western blot and include a human ovarian or oocyte lysate as a positive control on the same membrane. If you observe a band near 48 kDa in mouse but not in a negative control tissue lacking oocytes, that supports specificity. Rat and other mammalian species are similarly predicted based on homology.

What sample preparation is best for detecting ovastacin by Western blot?

Ovastacin is expressed exclusively in oocytes and stored in cortical granules, so whole ovary lysates will be dilute unless you enrich for oocytes. If working with ovarian tissue, consider cumulus-oocyte complexes or denuded oocytes for concentrated signal. Use a standard RIPA or NP-40 lysis buffer with protease inhibitors; ovastacin is a metalloprotease so EDTA in your buffer will inhibit autocatalytic activity. Load 20 to 50 micrograms total protein per lane. For zona pellucida samples post-fertilization, you may need to solubilize zonae separately to detect cleaved ZP2 and residual ovastacin.

What is the recommended starting dilution for ovastacin Western blot?

We recommend a starting dilution of 1 to 1000 for Western blot, which works well with standard chemiluminescent detection when loading 20 to 50 micrograms of oocyte-enriched lysate. If your sample is whole ovary or low in oocyte content, you may need to increase antibody concentration to 1 to 500 or enrich your starting material. Titrate against your specific lysate; polyclonal antibodies from rabbit typically give robust signal in this range. Use five percent milk or BSA in TBS-T for blocking. Expect clean bands with minimal background given the restricted expression pattern of ovastacin.

What controls should I include when using this ovastacin antibody?

Include a positive control rich in oocytes: human cumulus-oocyte complexes, GV or MII stage oocytes, or ovarian lysate are ideal. Negative controls should lack oocytes entirely, such as liver, spleen, or somatic cell lines, since ASTL expression is oocyte-restricted. For functional studies, compare unfertilized oocytes with fertilized embryos at the two-cell stage; ovastacin is secreted from cortical granules after fertilization, so localization shifts. A no-primary-antibody control will rule out secondary antibody artifacts. If available, an ASTL knockout or knockdown sample provides definitive specificity confirmation.

How should I store the ovastacin antibody and what is its shelf life?

Store the antibody at minus 20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100-microgram pack size is suitable for aliquoting into 10 to 20 microliter volumes depending on your typical experiment frequency. Polyclonal rabbit antibodies from Triple Point Biologics remain stable for at least two years under these conditions. If you need working stock for frequent use, keep a small aliquot at four degrees Celsius with 0.02 percent sodium azide for up to one month. Do not store diluted antibody in blocking buffer for more than one week.

Does ovastacin have isoforms or splice variants I should be aware of?

Ovastacin is encoded by a single-copy gene, ASTL, and the primary transcript produces a 431-amino-acid protein. Alternative splicing has not been widely reported for ovastacin in the literature, and the UniProt entry Q6HA08 lists one canonical sequence. However, you may observe post-translational modifications: ovastacin is synthesized as a zymogen with a pro-domain that is likely cleaved upon secretion from cortical granules. The antibody targets a broad epitope range, so it should detect both pro-ovastacin and the mature active form. Glycosylation variants may cause band heterogeneity on Western blot as noted earlier.