Anti-PC-7 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for PC-7 on a Western blot?

PC-7 migrates as a full-length precursor of approximately 87-90 kDa and a mature, autocatalytically processed form around 66-68 kDa. You may observe both species simultaneously depending on cell type and lysis conditions, as PC-7 undergoes activation in the trans-Golgi network. Some tissues or overexpression systems show predominantly the mature form. Under reducing conditions, the prodomain (cleaved during autocatalytic maturation) is typically not retained in the complex, so you will see single bands rather than doublets at lower molecular weights. Always include a positive control lysate known to express PC-7 for band assignment.

Does this PC-7 antibody distinguish between proPC-7 and mature PC-7?

This polyclonal antibody is raised against a region spanning residues that are present in both the zymogen and mature forms of PC-7, so it will detect both proPC-7 (approximately 87-90 kDa) and the autocatalytically processed mature enzyme (approximately 66-68 kDa). If your experimental question requires distinguishing activation state, run samples under reducing conditions and assess the relative intensities of the two bands. Inhibition of furin-like convertases or expression in secretion-deficient cells may shift the ratio toward the proform. The antibody does not selectively immunoprecipitate one form over the other.

What dilution should I start with for Western blot of PC-7?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this antibody. Because PC-7 is often expressed at moderate to low endogenous levels, you may need to optimize between 1:500 and 1:2000 depending on your sample source and ECL sensitivity. Use 20-50 micrograms of total protein per lane from whole-cell lysates. Membrane-enriched fractions will yield stronger signal since PC-7 localizes to the trans-Golgi network. Overnight incubation at four degrees Celsius often improves signal-to-noise ratio compared to one-hour room-temperature incubations for low-abundance targets like PC-7.

Is this antibody validated for mouse and rat PC-7 or only human?

This antibody has been validated experimentally for human PC-7. Reactivity with mouse and rat is predicted based on sequence homology in the immunogen region but has not been verified in-house. Human and mouse PC-7 share approximately 85 percent identity at the protein level, with strong conservation in the catalytic and P domains. If you are working with rodent samples, we recommend running a pilot blot with a known PC-7-positive tissue such as liver or kidney lysate alongside a knockout or siRNA knockdown control to confirm specific detection before proceeding with experimental samples.

What sample preparation is best for detecting endogenous PC-7 in cell lysates?

PC-7 is a type I transmembrane protein anchored in the trans-Golgi network, so use lysis buffers containing nonionic detergent such as one percent Triton X-100 or 0.5 percent NP-40 in Tris-buffered saline with protease inhibitors. RIPA buffer works well for total lysates. Avoid freeze-thaw cycles that can degrade membrane-associated proteins. Because PC-7 undergoes autocatalytic processing, include a serine protease inhibitor cocktail if you want to preserve the proform, though this is not necessary for standard detection. Membrane-enriched fractions prepared by differential centrifugation will concentrate PC-7 and improve detection compared to whole-cell lysates.

Can I use this PC-7 antibody for immunohistochemistry on paraffin sections?

Triple Point Biologics antibodies are routinely validated for immunohistochemistry, and this PC-7 antibody is suitable for IHC applications. For paraffin-embedded tissues, perform heat-induced epitope retrieval using citrate buffer pH 6.0 or Tris-EDTA pH 9.0; test both to determine which gives better signal in your tissue type. Start with a 1:100 to 1:200 dilution and titrate as needed. PC-7 expression is often strongest in secretory tissues such as liver, intestine, and endocrine glands. Include a no-primary-antibody control and, ideally, a PC-7 knockout or siRNA-treated tissue section to confirm staining specificity.

How should I store the PC-7 antibody and how long is it stable?

Store the antibody at minus twenty 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 10-20 microliter volumes depending on your typical Western blot usage at 1:1000 dilution. For short-term use within two to four weeks, the working aliquot can be kept at four degrees Celsius with 0.02 percent sodium azide as preservative if not used for applications incompatible with azide. Properly stored aliquots remain stable for at least twelve months. Centrifuge briefly before use if you observe any precipitate after thawing.

What positive control should I use to validate PC-7 detection?

HeLa, HepG2, and HEK293 cells all express detectable endogenous PC-7 and serve as accessible positive controls for Western blot. Liver and kidney tissue lysates are good choices for tissue-derived controls, as PC-7 is constitutively expressed in these organs. If you have access to a PC-7 overexpression plasmid, transiently transfected HEK293T lysate provides a strong positive control showing both pro- and mature forms. For a negative control, use a lysate from cells treated with PC-7-targeted siRNA for 48-72 hours, or compare signal between wild-type and PC-7 knockout cell lines if available in your system.