Anti-Carboxypeptidase E Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Carboxypeptidase-E on Western blot?

Carboxypeptidase-E migrates at approximately 53 kDa on reducing SDS-PAGE, corresponding to the mature enzyme after signal peptide cleavage from the 476-amino acid precursor. In neuroendocrine cell lysates, you may also observe a higher MW band near 55-60 kDa representing glycosylated forms, as CPE contains N-glycosylation sites. Some preparations show a faint band around 48 kDa, likely a processed form. The predominant signal should be in the 53 kDa range when using whole-cell lysates from CPE-expressing tissues like PC12 cells or pituitary.

Which tissues or cell lines serve as good positive controls for CPE antibody validation?

Neuroendocrine tissues and cell lines provide the strongest positive controls given CPE's tissue distribution. Rat or mouse pituitary lysate, adrenal medulla, or hypothalamic extracts are excellent primary tissue sources. For cell lines, PC12 pheochromocytoma cells, AtT-20 corticotroph cells, and INS-1 or MIN6 pancreatic beta cells all express abundant endogenous CPE. Human neuroblastoma lines like SH-SY5Y also work. Avoid fibroblasts or non-secretory epithelial lines as negative controls, since CPE expression is largely restricted to the regulated secretory pathway.

Is this CPE antibody validated for mouse or rat samples, or only human?

This antibody is validated specifically for human CPE. However, cross-reactivity with mouse and rat is predicted based on high sequence homology in the immunogen region—human CPE shares approximately 85-90% identity with rodent orthologs across most epitope domains. Many researchers successfully use this antibody for rodent brain and pituitary lysates at similar dilutions, but you should verify specificity in your system with appropriate positive controls. We recommend testing at the suggested 1:1000 dilution initially and confirming band size matches expected MW.

What starting dilution should I use for immunohistochemistry with this CPE antibody?

For immunohistochemistry on paraffin-embedded sections, start with a 1:100 to 1:200 dilution and optimize from there based on signal intensity and background in your specific tissue. Antigen retrieval is typically required—citrate buffer (pH 6.0) heat-induced epitope retrieval works well for CPE in neuroendocrine tissues. For frozen sections, 1:200 to 1:500 often provides good signal. Expected staining pattern includes cytoplasmic puncta corresponding to secretory vesicles in CPE-positive cells. Pituitary, pancreatic islets, and adrenal medulla sections serve as useful optimization tissues.

Does Carboxypeptidase-E have multiple isoforms I should know about?

CPE exists primarily as a single dominant isoform in most neuroendocrine tissues, though alternative splicing has been reported. The main functional form is the membrane-associated enzyme localized to secretory vesicles. A soluble secreted form also exists following cleavage of the C-terminal transmembrane-retention domain. Additionally, some studies describe a CPE-ΔN variant with altered sorting properties. On Western blot, you will typically detect the major 53 kDa form; additional bands may represent glycosylation variants or proteolytic processing rather than true splice isoforms. The antibody recognizes the core catalytic domain shared across these forms.

What sample preparation considerations are important for detecting CPE?

CPE is a vesicle-associated protein, so complete cell lysis is important—use RIPA or similar buffer with detergent rather than mild non-ionic detergent alone. Include protease inhibitors to prevent degradation, particularly if working with tissue homogenates rich in proteases. For membrane-bound CPE, ensure adequate solubilization; sonication or freeze-thaw cycles can improve yield. Load 20-30 µg total protein per lane for most neuroendocrine cell lysates. If signal is weak, verify your sample actually expresses CPE, as non-neuroendocrine cells may have very low or undetectable levels.

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

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise polyclonal antibody performance. The lyophilized or glycerol-stabilized formulation typically remains stable for at least 12 months under these conditions. Once reconstituted or thawed, working aliquots can be kept at 4°C for up to one month for frequent use. Do not store diluted antibody in working solutions. If you notice increased background or reduced signal intensity over time, prepare a fresh aliquot. Adding 0.02% sodium azide as preservative can extend 4°C storage life.

Can this antibody distinguish between catalytically active and inactive CPE?

No, this antibody recognizes CPE protein by linear or conformational epitopes independent of catalytic activity status. It cannot distinguish between enzymatically active CPE and inactive forms that may result from loss of the essential zinc cofactor, oxidation, or mutation. If you need to assess CPE enzymatic activity specifically, you must use functional enzyme assays with appropriate CPE substrates like dansyl-Phe-Ala-Arg-Arg. The antibody is useful for quantifying total CPE protein levels and localization but does not report on catalytic competence. Combine Western blot data with activity assays for complete characterization.