Anti-PC-6 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for PC-6 on a Western blot?

The mature PC-6 protein runs at approximately 105-115 kDa on reducing SDS-PAGE, corresponding to the 969-residue catalytically active form after removal of the signal peptide and pro-domain. You may also observe higher molecular weight bands around 130-140 kDa representing the full-length zymogen before autocatalytic cleavage. Because PC-6 is a secreted glycoprotein that traffics through the constitutive secretory pathway, glycosylation status and post-translational modifications can shift the apparent molecular weight. Media and cell-surface fractions typically yield stronger signal than cytosolic lysates.

What is the recommended starting dilution for this PC-6 antibody in Western blot?

Start at 1:1000 dilution in blocking buffer for Western blot, as validated by Triple Point Biologics. This polyclonal antibody has been optimized for detection of human PC-6 at that working concentration. Depending on your expression level and sample type, you may titrate between 1:500 and 1:2000 to balance signal intensity and background. Secreted PC-6 in conditioned media or extracellular matrix fractions often requires less antibody than whole-cell lysates because the protein concentrates in those compartments. Always include a PC-6-expressing positive control tissue or cell line to confirm optimal working dilution.

Does this antibody cross-react with other proprotein convertases like furin or PC5?

This rabbit polyclonal was raised against a PC-6-specific immunogen, minimizing cross-reactivity with related subtilisin/kexin family members such as furin, PC1, PC2, PC5, and PC7. However, because these convertases share significant sequence homology in their catalytic domains, you should validate specificity in your system. Run parallel blots with lysates known to express different PCs, or use siRNA knockdown of PC-6 to confirm that the band of interest disappears. Furin is ubiquitously expressed and runs at approximately 90-100 kDa, slightly lower than PC-6, which aids in distinguishing the two on Western blots.

Which human tissues and cell lines express PC-6 at detectable levels?

PC-6 shows a more restricted distribution than furin. Highest expression occurs in neuroendocrine tissues including pituitary, adrenal medulla, and enteroendocrine cells of the gut. Non-neuroendocrine sites include liver hepatocytes, ovarian granulosa cells, and the developing heart. For positive controls, consider AtT-20 pituitary cells, HepG2 hepatoma cells, or ovarian cancer lines such as SKOV3. Negative controls include most hematopoietic cells and fibroblasts, which express furin but little or no PC-6. Conditioned medium from PC-6-expressing cells is often the best source because the protein is secreted and can be concentrated.

Can I use this PC-6 antibody on mouse or rat samples?

This antibody is validated for human PC-6. Cross-reactivity with mouse and rat PC-6 is predicted based on sequence homology, typically above eighty-five percent in the catalytic and cysteine-rich domains, but has not been experimentally confirmed by Triple Point Biologics. If you plan to work with rodent tissues or cells, run pilot Western blots using lysates from mouse liver or rat pituitary as positive controls alongside a human sample. Expect the same molecular weight range, approximately 105-115 kDa for the mature form. Titrate the antibody and validate band identity with a blocking peptide or knockdown if available.

What sample preparation steps are critical for detecting secreted PC-6?

Because PC-6 is secreted and associates with the extracellular matrix via its C-terminal cysteine-rich domain, collect conditioned culture medium and consider enriching cell-surface or matrix fractions. For Western blot, concentrate serum-free conditioned medium by ultrafiltration or precipitation to increase sensitivity. Include protease inhibitors during lysis and avoid repeated freeze-thaw cycles, which can degrade the protease. Run samples under reducing conditions to fully denature the protein. For immunohistochemistry, PC-6 localizes to secretory granules and the extracellular space, so antigen retrieval with citrate buffer often improves epitope accessibility in fixed tissues.

How should I store this PC-6 antibody and what is the expected shelf life?

Store the antibody at minus twenty degrees Celsius in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100-microgram pack size is sufficient for approximately one hundred Western blots at 1:1000 dilution. For long-term storage beyond one year, minus eighty degrees is preferable. Once thawed, an aliquot remains stable at four degrees Celsius for up to one month if supplemented with sodium azide or another preservative. Do not store diluted antibody in blocking buffer for more than one week. Triple Point Biologics has manufactured proteinase antibodies since 1994, and this polyclonal typically retains activity for at least two years under proper storage.

What are good positive and negative controls for PC-6 Western blots?

Use lysates or conditioned medium from a PC-6-expressing cell line such as AtT-20 mouse pituitary cells, HepG2 hepatoma, or human ovarian cancer lines as positive controls. Human liver tissue lysate is also a reliable choice. For a negative control, use HEK293 or NIH3T3 fibroblasts, which express furin but minimal PC-6, to assess antibody specificity. A blocking peptide corresponding to the immunogen can confirm that the band is PC-6-specific; pre-incubate the antibody with peptide and verify loss of signal. If available, PC-6 knockdown or knockout lysates provide the strongest negative control to rule out off-target binding.