Anti-PC-4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for PC-4 on a Western blot?

PC-4 migrates at approximately 84–88 kDa on reducing SDS-PAGE, corresponding to the mature, catalytically active form after autocatalytic removal of the N-terminal prosegment. The full-length precursor (proPC-4) is 755 residues and predicted at roughly 86 kDa before cleavage, but you will typically detect the processed species in lysates from expressing tissues. Post-translational glycosylation can shift the apparent mass upward; placental and testicular samples may show slight mobility differences. Start with 1:1000 dilution and 20–50 µg total protein per lane for initial detection.

Which tissues or cell lines express PC-4 at detectable levels?

PC-4 expression is largely restricted to the reproductive tract. In males, testicular germ cells—particularly round and elongating spermatids—show robust expression. In females and pregnancy, placental trophoblast is the principal site. Most somatic cell lines (HeLa, HEK293, A549) do not express endogenous PC-4, making them poor positive controls. For human samples, term placenta homogenate or enriched human testis lysate serve as reliable positive controls. Mouse testis is a useful cross-species control given the predicted reactivity, though validation in your specific model is recommended.

Does this antibody cross-react with other proprotein convertases like furin or PCSK5?

PC-4 shares significant sequence homology with furin (PCSK3) and PCSK5 within the catalytic domain, but this polyclonal was raised against a longer stretch (residues 46–460 region) that includes divergent regulatory and P domains. In our hands, no cross-reactivity with furin, PCSK1, or PCSK2 was observed in Western blots of lysates co-expressing multiple convertases. However, PCSK5 shares closer homology; if your sample expresses PCSK5, include a knockout or RNAi-depleted control lane to confirm band identity. Immunoprecipitation or siRNA knockdown remains the definitive specificity check for any PCSK family member.

What dilution should I start with for immunohistochemistry on paraffin sections?

For IHC on formalin-fixed paraffin-embedded (FFPE) human testis or placenta, start at 1:200 dilution after antigen retrieval in citrate buffer, pH 6.0, for 20 minutes. PC-4 epitopes are moderately resistant to fixation; we routinely retrieve with pressure-cooker or microwave methods. Testis sections show strong cytoplasmic and acrosomal staining in spermatids; trophoblast cytoplasm stains diffusely. Omit primary antibody on an adjacent section as a negative control. If background is high, increase dilution to 1:400 and extend blocking time with normal goat serum.

Is this PC-4 antibody validated in mouse and non-human primate samples?

The antibody is validated in human samples. Mouse and non-human primate (macaque, marmoset) are listed as predicted cross-reactive based on sequence homology in the immunogen region, which exceeds 80 percent identity. We have preliminary Western blot data showing a band near 85 kDa in mouse testis lysate, consistent with mouse Pcsk4, but full titration and blocking controls have not been completed. If you work with rodent or primate models, plan a dilution series (1:500 to 1:2000) and include a Pcsk4-null mouse lysate or RNAi-treated sample to confirm specificity before committing to large-scale experiments.

How should I store the antibody and what is the shelf life after reconstitution?

The lyophilized antibody is stable at –20 °C for at least two years from the manufacturing date printed on the vial label. Reconstitute the 100 µg aliquot in 100 µl sterile phosphate-buffered saline (final concentration 1 mg/ml); gently invert—do not vortex. After reconstitution, aliquot into single-use volumes (10–20 µl) to avoid repeated freeze-thaw cycles, and store at –20 °C or –80 °C. Under these conditions, activity remains stable for at least six months. For routine use, a working dilution in PBS with 0.02 percent sodium azide can be kept at 4 °C for two weeks.

Why do I see no signal in HEK293 or HeLa lysates on Western blot?

HEK293 and HeLa cells do not express endogenous PC-4; the protease is transcriptionally silent outside reproductive tissues. You will not detect a band unless you transfect a PC-4 expression vector. For positive-control lysates, use human term placenta (available commercially), enriched human or mouse testis homogenate, or a mammalian cell line stably transfected with PCSK4 cDNA. If you are studying PC-4 regulation or substrate cleavage in a heterologous system, confirm transgene expression by RT-PCR in parallel, then validate the antibody signal against a vector-only or untransfected control lane.

Can I use this antibody to detect PC-4 in human seminal plasma or follicular fluid?

PC-4 is predominantly membrane-associated or intracellular within germ cells and trophoblast; secretion into seminal plasma or follicular fluid occurs at very low levels, if at all. Published reports have not convincingly demonstrated soluble PC-4 in these biofluids by Western blot. If you wish to attempt detection, concentrate seminal plasma at least tenfold by ultrafiltration (10 kDa cutoff) and load 50 µg per lane, but expect weak or absent signal. For functional studies of PC-4 activity in extracellular compartments, enzymatic assays with fluorogenic substrates may be more sensitive than immunoblotting.