Anti-PCSK5 / PC-5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for PC-5 on a Western blot?

Full-length PCSK5 is 1860 amino acids with a predicted molecular weight around 206 kDa. However, PC-5 undergoes autocatalytic maturation and proteolytic processing, so you will typically observe multiple bands on Western blot. The mature secreted form runs at approximately 130-140 kDa after removal of the prodomain. Depending on cell type and secretory pathway activity, you may also detect the prodomain fragment around 75 kDa or intermediate processing forms. Post-translational modifications including glycosylation can shift apparent molecular weight. Always include a positive control lysate from cells known to express PCSK5 when interpreting your blot.

What is the recommended starting dilution for Western blot with this PC-5 antibody?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal anti-PC-5 antibody. This dilution has been validated in our hands using human cell lysates. Depending on your expression levels and detection system sensitivity, you may need to optimize between 1:500 and 1:2000. Because PC-5 is a secreted protease, conditioned media from expressing cells often yields stronger signal than whole-cell lysates. Use 20-30 micrograms total protein per lane as a starting point. Standard blocking with five percent non-fat milk in TBST for one hour at room temperature works well.

Does this antibody detect both PC-5A and PC-5B isoforms?

PCSK5 generates two major splice variants, PC-5A and PC-5B, that differ in their C-terminal domains. PC-5A contains a cysteine-rich domain and is primarily secreted, while PC-5B has a transmembrane domain and remains mostly cell-associated. The immunogen region for this polyclonal antibody falls within the shared N-terminal catalytic domain common to both isoforms, so it should recognize both PC-5A and PC-5B variants. The molecular weight difference between isoforms is modest, approximately 10-15 kDa, and may not always resolve clearly on standard SDS-PAGE gels depending on running conditions and processing state.

Will this PC-5 antibody cross-react with other proprotein convertases like furin or PC7?

The subtilisin-like proprotein convertase family shares significant sequence homology in their catalytic domains, particularly furin, PC5, PACE4, and PC7. While this antibody was raised against PC-5-specific sequences, some degree of cross-reactivity with closely related family members is possible with polyclonal sera. If you are working with samples expressing multiple convertases, consider testing furin-null or PC5-specific knockout cell lines as negative controls. Alternatively, siRNA knockdown of PCSK5 followed by Western blot can confirm band identity. The expected molecular weights differ somewhat among family members, which aids interpretation, but definitive identification may require immunoprecipitation followed by mass spectrometry.

What positive control cell line should I use for PC-5 antibody validation?

PCSK5 is broadly expressed but levels vary considerably by tissue and cell type. Human placental tissue or trophoblast cell lines such as BeWo or JEG-3 express high levels of PC-5 due to its essential role in pregnancy establishment, making them excellent positive controls. HepG2 hepatocellular carcinoma cells and HT-1080 fibrosarcoma cells also express detectable PCSK5. Because PC-5 is secreted, harvest both cell lysate and conditioned media; the latter often shows stronger signal. For negative controls, consider using lysates from cells treated with PCSK5-targeting siRNA for 48-72 hours to demonstrate antibody specificity against your target band.

Can I use this antibody for immunohistochemistry on human tissue sections?

This antibody is validated for Western blot at 1:1000 dilution and our antibodies are typically compatible with immunohistochemistry applications, though optimal conditions for IHC will require empirical testing. For paraffin-embedded human tissue, start with antigen retrieval using citrate buffer pH 6.0 at sub-boiling temperature for 20 minutes. Begin antibody titration at 1:100 to 1:200 dilution and optimize from there. PC-5 expression is prominent in placenta, liver, kidney, and intestinal epithelium. Because PC-5 is secreted, you may observe diffuse extracellular staining in addition to cytoplasmic signal. Include appropriate isotype controls and known PCSK5-negative tissue to assess background.

Does this PC-5 antibody work with mouse or rat samples?

This antibody is validated for use with human samples. Reactivity with mouse or rat PC-5 is predicted based on sequence homology but has not been experimentally confirmed in our laboratory. Mouse and rat PCSK5 share approximately 85-90 percent amino acid identity with human PCSK5 in the catalytic domain, suggesting cross-reactivity is likely. If working with rodent samples, we recommend testing the antibody at the standard 1:1000 dilution on lysates from mouse liver or placenta, which express PCSK5 at detectable levels. Confirm band identity by comparing molecular weight to predicted species-specific sizes and consider peptide competition or knockout tissue controls for validation.

How should I store this PC-5 antibody and what is the expected shelf life?

Store the PC-5 antibody at minus 20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce antibody titer and increase aggregation. The antibody is supplied at 100 micrograms total and is stable for at least 12 months when stored properly at minus 20 degrees. For frequent use, keep a working aliquot at 4 degrees for up to one month; add sodium azide to 0.02 percent final concentration as preservative if desired, but omit azide if using HRP-conjugated secondary antibodies. Avoid storing in frost-free freezers due to temperature fluctuations. Centrifuge briefly before use if any precipitate forms after thawing.