Anti-Carboxypeptidase A6 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CPA6 on Western blot?

CPA6 migrates at approximately 48-50 kDa on reducing SDS-PAGE, corresponding to the mature secreted form of the 437-residue enzyme after signal peptide cleavage. The predicted molecular weight from the full-length sequence is higher, but the processed extracellular form predominates in most lysates. You may observe additional higher molecular weight bands around 55-60 kDa in some tissues, potentially representing glycosylated variants or incompletely processed forms. Brain and neuronal samples typically show the cleanest single band. If working with conditioned media or extracellular matrix preparations, expect the mature processed form.

What starting dilution should I use for CPA6 Western blot?

Start at 1:1000 dilution in 5% non-fat milk or BSA in TBST for Western blot applications, as recommended for this polyclonal. This dilution has been validated with human tissue and cell lysates. Depending on CPA6 expression levels in your sample, you may need to optimize between 1:500 and 1:2000. Brain tissue and neuronal cell lines generally express CPA6 at moderate levels and work well at 1:1000. For samples with lower expression, particularly non-neuronal tissues, consider starting at 1:500. Overnight incubation at 4°C often improves signal over a one-hour room temperature incubation.

Does this antibody cross-react with other carboxypeptidase A family members?

CPA6 shares sequence homology with other M14A family carboxypeptidases, particularly CPA1-5, which raises potential cross-reactivity concerns. This polyclonal has been validated specifically against human CPA6, but we have not systematically tested cross-reactivity with all CPA family members. CPA4 shares the highest structural similarity and could potentially cross-react depending on the epitope region. If you are working with tissues or cells that co-express multiple CPA isoforms, consider including CPA1 or CPA4 knockout lysates as specificity controls, or validate band identity by siRNA knockdown of CPA6. Peptide competition assays can also confirm specificity.

Will this rabbit polyclonal work with mouse or rat samples?

The antibody has been validated against human CPA6 only. Mouse and rat CPA6 orthologs share approximately 75-80% sequence identity with human CPA6, which suggests potential cross-reactivity, but this remains predicted rather than validated. If you plan to use mouse or rat samples, we recommend testing the antibody at the standard 1:1000 dilution first, using brain tissue lysate as a positive control since CPA6 is well-expressed in rodent CNS. Include a known human positive control in parallel to compare band patterns and molecular weight. We cannot guarantee performance in non-validated species without empirical testing in your hands.

What are good positive and negative control tissues for CPA6?

Human brain tissue, particularly cerebellum and hippocampus, serves as a strong positive control given CPA6's role in neuropeptide processing. Neuroblastoma or glioma cell lines also typically express detectable CPA6. For secreted protein detection, collect conditioned media from neuronal cultures after 48-72 hours. As a negative control, CPA6 expression is low to absent in most hematopoietic cells and skeletal muscle. For definitive negative controls, use lysate from cells transfected with CPA6-targeting siRNA alongside scrambled control. This approach confirms that your observed band specifically represents CPA6 rather than a cross-reacting protein or non-specific binding artifact.

Can I use this antibody for immunohistochemistry on brain sections?

this antibody is validated for Western blot; IHC validation is in progress applications. For paraffin-embedded brain sections, perform antigen retrieval using citrate buffer pH 6.0 with heat-induced epitope retrieval. Start with a 1:100 to 1:200 dilution for IHC, which is more concentrated than the Western blot recommendation due to tissue fixation masking epitopes. CPA6 localizes to the extracellular matrix and neuropil in brain tissue, so expect diffuse extracellular staining patterns rather than sharp intracellular localization. Include sections from CPA6-low tissues as negative anatomical controls. Blocking with 10% normal goat serum reduces background with this rabbit polyclonal when using goat anti-rabbit secondary antibodies.

How should I prepare samples to detect secreted CPA6?

Since CPA6 is secreted into the extracellular space, collect conditioned media from cultured cells or use serum and cerebrospinal fluid samples. For conditioned media, culture cells to 70-80% confluence, wash twice with PBS, then incubate in serum-free media for 24-48 hours. Centrifuge media at 2000×g to remove debris, then concentrate 10-20-fold using centrifugal filters with 10 kDa cutoff. Add protease inhibitors immediately after collection to prevent CPA6 autoproteolysis or degradation by other proteases. For tissue lysates, include extracellular matrix extraction steps or use gentle homogenization buffers that preserve secreted proteins rather than strictly intracellular lysis conditions.

What is the recommended storage condition and shelf life for this antibody?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation of polyclonal IgG. The antibody is supplied in a glycerol-containing buffer that remains liquid at -20°C, allowing you to remove small volumes without fully thawing if necessary, though aliquoting remains preferable. Under these conditions, the antibody typically maintains activity for at least 12 months from receipt. Avoid storage at 4°C for extended periods beyond one month. If you observe increased background or decreased signal over time, this usually indicates antibody degradation or aggregation requiring a fresh aliquot or new vial.