Anti-Carboxypeptidase-A2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CPA2 on a Western blot?

The mature, catalytically active form of carboxypeptidase A2 runs at approximately 34–35 kDa, corresponding to the processed enzyme after removal of the N-terminal activation peptide. In pancreatic lysates or culture supernatants from acinar cells, you may also detect the inactive zymogen form (procarboxypeptidase A2) at roughly 46–48 kDa if cleavage by trypsin has not yet occurred. The size difference reflects the propeptide segment. If you observe both bands, it indicates a mixed population of zymogen and mature enzyme, which is common in samples collected before or during secretory activation.

Does this antibody cross-react with CPA1 or other carboxypeptidases?

This antibody is raised against human CPA2 and has been validated specifically for CPA2 detection. While CPA1 and CPA2 share structural homology as members of the M14 metallocarboxypeptidase family, their sequence identity is approximately 50–60 percent, and epitope recognition typically confers specificity. We have not observed cross-reactivity with CPA1 in validated human pancreatic lysates, but formal cross-reactivity testing against recombinant CPA1, CPA4, or CPA5 has not been completed. If you are working in a system expressing multiple CPA isoforms, include isoform-specific controls or knockdown samples to confirm signal identity.

What starting dilution should I use for Western blot with pancreatic lysates?

We recommend starting at 1:1000 for Western blot of human pancreatic tissue or cell lysates, where CPA2 is abundantly expressed. Because CPA2 is a secreted enzyme stored in zymogen granules, expression is high in acinar cells, and this dilution typically yields clean, specific bands with standard chemiluminescent detection. For non-pancreatic samples or low-abundance systems, you may need to increase antibody concentration to 1:500 or 1:750. Conversely, if background is high, titre up to 1:2000. We suggest running a dilution series with your specific sample type to establish optimal signal-to-noise for your detection system.

Can I use this antibody to detect mouse or rat CPA2?

The antibody has been validated against human CPA2. Mouse and rat CPA2 share approximately 80–85 percent amino acid identity with the human orthologue, and cross-reactivity is predicted but not experimentally validated by us. If you are working with rodent samples, we recommend treating this as a pilot experiment: run positive controls (human pancreatic lysate) alongside your mouse or rat samples on the same blot. Many researchers report successful detection of rodent CPA2 with antibodies raised against the human protein, but band intensity and background may differ. Confirm the expected molecular weight and consider sequence alignment of your target epitope region if results are inconclusive.

Is CPA2 expressed in tissues other than pancreas?

CPA2 is predominantly expressed in the exocrine pancreas, where it is synthesized by acinar cells, stored as a zymogen, and secreted into pancreatic juice. Expression in other tissues is generally low or absent under normal conditions. Some studies report low-level expression in certain tumour types or during pancreatic injury and inflammation, but these contexts are not routine. If you are investigating non-pancreatic samples and detect a band at the expected molecular weight, validate specificity with a blocking peptide or CPA2 knockdown control, as off-target binding becomes more likely when the cognate antigen is scarce.

What sample preparation steps are critical for preserving CPA2 signal?

Because CPA2 is a secreted zymogen stored in granules, gentle lysis conditions that preserve membrane-bound compartments can improve yield. Use protease inhibitor cocktails that include metalloprotease inhibitors, since CPA2 itself is a zinc-dependent enzyme and may be subject to proteolytic processing or degradation during lysis. Avoid repeated freeze-thaw cycles, which can aggregate or degrade both zymogen and mature forms. If you are detecting the secreted enzyme in conditioned medium or pancreatic juice, concentrate samples by acetone precipitation or ultrafiltration before loading to improve signal intensity, as dilute secreted protein may fall below detection threshold at standard loading volumes.

How should I store the antibody, and what is the expected shelf life?

Store the antibody at –20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. Once thawed, an aliquot can be held at 4°C for up to two weeks if sodium azide (0.02–0.05 percent final) is present as a preservative; consult the product datasheet for formulation details. Do not store diluted antibody in blocking buffer for more than 24 hours, as carrier proteins can promote bacterial growth and destabilize the immunoglobulin. Under proper storage conditions, the antibody typically retains activity for at least 12 months from the date of receipt. Verify lot-specific expiry on the vial label.

What positive and negative controls should I include when using this CPA2 antibody?

Human pancreatic tissue lysate or lysate from a pancreatic cell line such as PANC-1 serves as a strong positive control, given the high endogenous expression of CPA2 in acinar cells. For a negative control, use lysate from a tissue or cell line with negligible CPA2 expression, such as HeLa or NIH-3T3 cells, to assess non-specific binding. If you have access to CPA2 knockdown or knockout cell models, these provide the most rigorous specificity control. Additionally, pre-incubating the antibody with a CPA2 blocking peptide before blotting can confirm that the observed band corresponds to CPA2 rather than a cross-reactive epitope.