Anti-PCOLCE2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for PCOLCE2 on Western blot?

PCOLCE2 (Q9UKZ9) has a predicted molecular weight of approximately 47 kDa based on its 415-amino acid sequence. However, PCOLCE2 is a secreted glycoprotein that undergoes N-glycosylation in the extracellular space, so the observed band typically migrates between 50-60 kDa depending on glycosylation state and cell type. If you see multiple bands in conditioned media or ECM-enriched lysates, consider glycosylation heterogeneity rather than degradation. Deglycosylation with PNGase F can confirm glycosylation if band identity is unclear. Start with the recommended 1:1000 dilution for human samples.

Does this PCOLCE2 antibody cross-react with PCOLCE1 or other ECM proteins?

PCOLCE2 shares approximately 50 percent sequence identity with PCOLCE1 (PCPE-1), particularly in the CUB and NTR domains, so cross-reactivity is a reasonable concern. This rabbit polyclonal is validated for human PCOLCE2; we have not formally tested cross-reactivity with PCOLCE1. If you work in systems where both paralogs are expressed, consider running lysates from PCOLCE1 or PCOLCE2 knockout cells as negative controls, or include recombinant PCOLCE1 protein to assess specificity by competitive blotting. Cross-reactivity with other ECM components like collagens or BMP1 has not been observed in our validation work.

What sample types work best for detecting PCOLCE2 with this antibody?

PCOLCE2 is a secreted protein that functions in the extracellular matrix, so conditioned media and ECM-enriched fractions yield stronger signals than whole-cell lysates. For Western blot, collect serum-free conditioned media after 24-48 hours, concentrate by TCA precipitation or ultrafiltration, and load 20-30 µg total protein. Tissue lysates from fibrotic liver, lung, or bone should be prepared with detergent buffers that solubilize ECM proteins. For IHC, PCOLCE2 localizes to connective tissue and sites of active collagen remodeling. Avoid over-fixation, which can mask epitopes in heavily cross-linked matrix.

What dilution should I start with for IHC and immunofluorescence?

The validated Western blot dilution is 1:1000, but IHC and immunofluorescence typically require higher antibody concentrations. Start with 1:100 to 1:200 for paraffin-embedded tissue sections and 1:200 to 1:400 for frozen sections or cultured cells. PCOLCE2 is an extracellular protein, so antigen retrieval is critical for FFPE tissues; citrate buffer (pH 6.0) at sub-boiling temperature for 20 minutes works well for most ECM antigens. Optimize on positive-control tissues such as developing bone, healing wounds, or fibrotic tissue where collagen remodeling is active. Titre against your specific sample type before committing to a large experiment.

Is this antibody predicted to work in mouse or rat samples?

This antibody is validated for human PCOLCE2. Mouse and rat PCOLCE2 share approximately 75-80 percent sequence identity with human, so cross-reactivity is plausible but not guaranteed. We have not formally validated reactivity in rodent samples. If you plan to use mouse or rat lysates, run a pilot blot alongside human control lysate to confirm a band at the expected size (50-60 kDa accounting for glycosylation). Consider using fibroblast conditioned media or fibrotic tissue lysates as positive controls, since PCOLCE2 expression is often low in normal homeostatic conditions. Contact us if you generate validation data in non-human species.

What positive and negative controls should I use for PCOLCE2 experiments?

Positive controls include conditioned media from primary human fibroblasts, osteoblasts, or chondrocytes, all of which secrete PCOLCE2 to support collagen maturation. Tissue lysates from fibrotic liver, lung, or skeletal tissue undergoing active remodeling also express PCOLCE2. Negative controls should include samples where PCOLCE2 is silenced (siRNA or CRISPR knockout) or tissue types with minimal ECM turnover. Recombinant human PCOLCE2 protein serves as a molecular weight reference and specificity control. Because PCOLCE2 enhances BMP1-mediated procollagen cleavage, functional assays can complement antibody-based detection to confirm biological activity in your system.

How should I store this antibody and what is the expected stability?

Store the antibody at -20°C in single-use aliquots to avoid freeze-thaw cycles, which can reduce titre and increase background. The 100 µg pack size is suitable for aliquoting into 10-20 µL volumes depending on your typical experiment scale. Once thawed, the antibody remains stable at 4°C for up to one month; add sodium azide (0.02-0.05 percent final concentration) as a preservative if you plan to store working dilutions. Avoid prolonged exposure to room temperature. Glycerol or BSA at 50 percent or 1-2 percent, respectively, can improve long-term stability but may require optimization if you observe high background in IHC applications.

Why is PCOLCE2 expression often low or undetectable in whole-cell lysates?

PCOLCE2 is a secreted glycoprotein that traffics through the ER and Golgi before release into the extracellular space, so intracellular pools are transient and typically low. Cells with high biosynthetic activity, such as fibroblasts under TGF-beta stimulation or osteoblasts during matrix mineralization, may show detectable intracellular signal, but conditioned media remains the preferred sample type. If you need to detect intracellular PCOLCE2, treat cells with brefeldin A or monensin to block secretion and accumulate the protein in the ER or Golgi. For Western blot of lysates, load at least 30-50 µg total protein and expose membranes longer to visualize low-abundance signal.