Anti-Carboxypeptidase-Z Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Full-length Carboxypeptidase-Z migrates at approximately 72-75 kDa on Western blot, corresponding to its 652-residue sequence plus glycosylation. Because CPZ is a secreted metallocarboxypeptidase with predicted N-glycosylation sites, the apparent molecular weight can vary depending on tissue source and post-translational modifications. If you observe bands at lower molecular weights, consider the possibility of proteolytic processing or alternative splice variants. For initial validation, include conditioned medium from CPZ-expressing cells or lysates from tissues with known high expression such as liver or kidney.

Does the Carboxypeptidase-Z antibody cross-react with other metallocarboxypeptidases?

This antibody is validated for human CPZ. The metallocarboxypeptidase family shares structural homology in the catalytic domain, so cross-reactivity with closely related enzymes such as carboxypeptidase A4 or carboxypeptidase B is possible but not systematically tested. The presence of the unique Frizzled-like cysteine-rich domain in CPZ may provide epitope specificity depending on the immunogen region. If you require absolute specificity in samples expressing multiple carboxypeptidases, confirm identity by running parallel blots with CPZ-knockdown lysates or include peptide competition controls to verify band specificity.

Can I use this antibody to detect Carboxypeptidase-Z in mouse or rat samples?

The antibody is validated for human CPZ. Cross-reactivity with mouse and rat orthologs is predicted based on sequence homology but not experimentally validated by Triple Point Biologics. Human CPZ shares approximately 85-90 percent identity with rodent orthologs, particularly in conserved functional domains. If working with rodent samples, start with the recommended 1:1000 dilution and be prepared to optimize. Include a positive control from human tissue or recombinant human CPZ alongside your rodent samples to assess comparative signal intensity and confirm the expected molecular weight.

What is the recommended starting dilution for Carboxypeptidase-Z antibody in Western blot?

Start at 1:1000 dilution for Western blot, which is the validated working dilution for this antibody. Because CPZ is a secreted protein localized to the extracellular space, signal intensity will depend heavily on your sample type. Conditioned medium and extracellular matrix preparations typically yield stronger signal than whole-cell lysates. If working with tissue homogenates, liver and kidney are good positive controls due to higher endogenous CPZ expression. For low-abundance samples, you may need to concentrate conditioned media or increase total protein load rather than increasing antibody concentration.

Should I use reducing or non-reducing conditions for Carboxypeptidase-Z Western blots?

Use reducing conditions with DTT or beta-mercaptoethanol for optimal results. The Frizzled-like cysteine-rich domain in CPZ contains multiple disulfide bonds, but Western blot detection of the denatured protein typically performs better under reducing conditions where the polypeptide backbone is fully linearized. Non-reducing conditions may alter migration or epitope accessibility depending on where the antibody binds. If you are specifically studying disulfide-linked complexes or CPZ interactions with binding partners, non-reducing gels are appropriate, but expect potentially different migration patterns and verify band identity with reducing gel comparison.

What are good positive and negative controls for Carboxypeptidase-Z experiments?

For positive controls, use human liver or kidney lysates, as CPZ shows relatively high expression in these tissues. Recombinant human CPZ is ideal when available. Conditioned medium from HEK293 cells transfected with CPZ expression constructs provides a clean positive control enriched for secreted protein. For negative controls, use lysates from CPZ-knockout cells if accessible, or pre-incubate the antibody with blocking peptide corresponding to the immunogen region. Because CPZ is secreted, cytosolic fractions should show minimal signal and can serve as an additional specificity control when compared to extracellular fractions.

How should I store the Carboxypeptidase-Z 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 antibody titre and increase aggregation. Once thawed, an aliquot can be kept at 4°C for up to one month for convenience during active experiments. Do not store diluted antibody in working solutions for extended periods; prepare fresh dilutions in blocking buffer for each experiment. If the antibody contains glycerol or other cryoprotectants, verify the formulation on the datasheet. Properly stored aliquots typically remain stable for at least one year from manufacture date, though activity should be monitored if older stocks show diminished signal.

Can I use this antibody for immunohistochemistry to localize Carboxypeptidase-Z in tissue sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry. For CPZ, expect extracellular and pericellular staining patterns consistent with its secreted localization. Antigen retrieval is generally recommended; citrate buffer at pH 6.0 with heat-induced epitope retrieval is a reasonable starting point, though optimal conditions depend on tissue fixation and embedding protocols. Start with 1:100 to 1:200 dilution and optimize based on signal-to-background ratio. Because CPZ may be diffusely distributed in extracellular matrix, include tissue known to express CPZ such as liver or use adjacent sections with and without primary antibody to confirm staining specificity.