Anti-Carboxypeptidase-O Rabbit Polyclonal Antibody

Frequently Asked Questions

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

The predicted molecular weight for full-length human Carboxypeptidase-O is approximately 42 kDa based on the 374-amino-acid sequence. Because CPO is a membrane-associated protein, you may observe additional higher-MW bands if post-translational modifications such as glycosylation are present in your sample. Running a positive control lysate from tissue or cells known to express CPO at the apical membrane will help confirm the expected migration pattern in your gel system. If you see multiple bands, consider whether your sample preparation retained membrane-associated forms versus soluble processed fragments.

What starting dilution should I use for Western blot with this CPO antibody?

We recommend starting at 1:1000 for Western blot, which has been validated with this antibody. Depending on CPO expression levels in your sample and your detection system sensitivity, you may need to optimize between 1:500 and 1:2000. Because CPO is a membrane protein with tissue-restricted expression patterns, samples with low endogenous CPO may require higher antibody concentration or longer exposure times. Always include a known positive control lysate to distinguish true signal from background, and consider membrane enrichment during sample preparation to improve detection.

Is this Carboxypeptidase-O antibody likely to cross-react with mouse or rat samples?

This antibody is validated for human CPO. Cross-reactivity with mouse or rat is predicted based on sequence homology but has not been experimentally validated. Human CPO shares significant conservation with rodent orthologs in the catalytic domain, so cross-reactivity is plausible. If you are working with mouse or rat samples, plan to empirically test the antibody at the recommended dilution range alongside a human positive control. Assess band patterns carefully, as non-specific binding can complicate interpretation without validated positive controls in your species of interest.

Can I use this antibody for immunofluorescence to detect CPO at the apical membrane?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence applications. Because CPO localizes to the apical cell membrane, proper sample preparation is critical. Use polarized epithelial cell models or tissue sections where apical domains are accessible. Ensure adequate permeabilization if detecting intracellular pools, though surface staining of non-permeabilized cells may work for apical CPO. Start with a dilution around 1:100 to 1:200 for IF and optimize based on signal intensity. Include cells or tissue with minimal CPO expression as a negative control to assess specificity of membrane staining.

What positive control tissue should I use for Carboxypeptidase-O Western blot?

CPO expression is enriched in tissues where apical membrane processing of peptides occurs, including kidney and intestinal epithelium. Kidney lysate is a practical positive control, as CPO participates in renal peptide processing. If you are studying CPO in a specific context, consider using lysate from polarized epithelial cell lines known to express CPO or tissue samples relevant to your research question. Membrane-enriched fractions will yield stronger signal than whole-cell lysates due to CPO's membrane association. Document your positive control source for reproducibility across experiments.

Does Carboxypeptidase-O have multiple isoforms I should be aware of on a blot?

Human CPO is encoded by a single gene, and the primary translated product is 374 amino acids. Alternative splicing has not been widely reported for CPO, so you should typically observe one major band corresponding to the full-length protein. However, membrane proteases can undergo proteolytic processing, and post-translational modifications may cause mobility shifts. If you observe multiple bands, consider whether your sample contains proteolytic fragments, differentially modified forms, or non-specific binding. Running samples with and without reducing agents can help distinguish disulfide-linked aggregates from monomeric CPO.

How should I store this antibody and how long will it remain stable?

Store the antibody at –20°C as per standard practice for polyclonal antibodies. Avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. For frequent use, consider preparing small aliquots at the first thaw. The antibody should remain stable for at least 12 months when stored properly at –20°C. If you notice decreased signal or increased background over time, this may indicate antibody degradation or contamination. Adding sodium azide to a final concentration of 0.02 to 0.05 percent can extend shelf life for liquid aliquots stored at 4°C for short-term use.

What sample prep considerations are important for detecting membrane-associated CPO?

Because CPO is a membrane-associated metallocarboxypeptidase, sample preparation significantly affects detection efficiency. Whole-cell lysates will contain CPO, but membrane-enriched fractions improve signal strength. Use lysis buffers containing non-ionic detergents such as Triton X-100 or NP-40 at 0.5 to 1 percent to solubilize membrane proteins effectively. Avoid harsh detergents like SDS during lysis, as these can denature CPO and complicate detection. Include protease inhibitors to prevent degradation during sample handling. If CPO is your target, sonication or mechanical disruption combined with detergent extraction yields better results than detergent alone.