Anti-PCOLCE (PCPE-1) Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for PCOLCE-1 on Western blot?

PCOLCE-1 migrates at approximately 48–55 kDa on reducing SDS-PAGE, slightly higher than the predicted 48 kDa for the 449-residue polypeptide due to N-linked glycosylation. You may observe a diffuse band or minor size heterogeneity reflecting variable glycosylation states, which is typical for this secreted glycoprotein. If you treat lysates with PNGase F or other deglycosylating enzymes, the band should sharpen and migrate closer to 48 kDa. Conditioned medium from fibroblasts or osteoblasts usually gives a cleaner signal than whole-cell lysates because PCOLCE-1 is secreted.

What dilution should I start with for PCOLCE-1 Western blot and IHC?

For Western blot, start at 1:1000 in 5% non-fat milk or BSA in TBST, which is the validated dilution for this antibody. For IHC on formalin-fixed paraffin-embedded sections, begin at 1:200 to 1:500 after antigen retrieval in citrate buffer, pH 6.0. Because PCOLCE-1 is a secreted protein, you may need to optimize blocking conditions and incubation time to reduce background in extracellular matrix-rich tissues. Always include a no-primary control and, if possible, a tissue known to express PCOLCE-1 such as skin or bone.

Does this antibody detect mouse or rat PCOLCE-1, or only human?

This antibody is validated for human PCOLCE-1. Cross-reactivity with mouse and rat is predicted based on sequence homology—human PCOLCE shares roughly 85–90% identity with mouse and rat orthologs across the CUB and netrin-like domains—but has not been experimentally confirmed by us. If you plan to use mouse or rat samples, we recommend running a positive control lysate from human cells in parallel and verifying band size and specificity. Titre the antibody starting at 1:500 to account for potential affinity differences.

Which cell types or tissues express high levels of PCOLCE-1 for positive controls?

PCOLCE-1 is most abundant in connective tissues undergoing active collagen synthesis. Primary human dermal fibroblasts, osteoblasts, and chondrocytes are excellent cellular sources; collect conditioned medium after 24–48 hours in serum-free media for the cleanest Western blot signal. At the tissue level, skin, bone, cartilage, and blood vessels show strong expression. Fetal or neonatal tissues generally express higher levels than adult tissues. Avoid using cell lines with low collagen turnover, such as epithelial carcinoma lines, unless you have RNA-seq or proteomic evidence of PCOLCE-1 expression.

Are there PCOLCE-1 isoforms or splice variants I should know about?

The major isoform is the canonical 449-residue protein encoded by the full-length PCOLCE transcript. A shorter splice variant, PCOLCE-2, is encoded by a separate gene and shares structural homology but is functionally distinct, so this antibody will not cross-react with PCOLCE-2. Within PCOLCE-1 itself, minor splice variants have been reported in the literature but are not well characterized and unlikely to be detected at significant levels in most tissues. Focus on the approximately 48–55 kDa band; additional bands may represent degradation products or non-specific binding rather than bona fide isoforms.

What sample preparation works best for detecting secreted PCOLCE-1?

Because PCOLCE-1 is secreted into the extracellular space, conditioned medium typically yields stronger signal than whole-cell lysate. Grow cells to 70–80% confluence, wash twice with PBS, then incubate in serum-free medium for 24–48 hours. Concentrate the conditioned medium 10- to 20-fold using a 10-kDa cutoff centrifugal filter to improve sensitivity. If working with tissue homogenates, include a protease inhibitor cocktail and consider separating the soluble and matrix-bound fractions; PCOLCE-1 may partition into both. For plasma or serum samples, dilute 1:5 to 1:10 before loading to avoid overwhelming the gel.

How should I store this PCOLCE-1 antibody and how long is it stable?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The stock solution typically contains glycerol or another cryoprotectant and remains stable for at least 12 months under these conditions. For day-to-day use, keep a working aliquot at 4°C for up to one month. Do not store diluted antibody in blocking buffer for more than a week, as preservative concentration drops and microbial contamination risk increases. If you see higher background or weaker signal over time, prepare a fresh aliquot.

What is the immunogen and epitope for this polyclonal PCOLCE-1 antibody?

This rabbit polyclonal was raised against a recombinant fragment corresponding to a large portion of the human PCOLCE-1 sequence, likely spanning residues within the CUB and netrin-like domains. The polyclonal nature means it recognizes multiple epitopes across that region, which generally improves sensitivity and resilience to post-translational modifications or mild denaturation. We have not mapped individual epitopes. Because it targets a broad immunogen, the antibody should detect both native and denatured PCOLCE-1, making it suitable for Western blot applications where antigen retrieval may alter epitope accessibility.