Anti-Carboxypeptidase A4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CPA4 on Western blot?

CPA4 is synthesized as a 421-amino-acid precursor with a predicted molecular weight of approximately 47 kDa. On reducing Western blots, you should expect a band near this size from cell lysates or conditioned media. Because CPA4 is a secreted metalloprotease that may undergo post-translational modification including glycosylation, the apparent molecular weight can shift slightly upward, sometimes appearing between 47-52 kDa depending on the tissue or cell type. If you observe additional lower bands, consider the possibility of proteolytic processing or degradation during sample preparation.

Does this antibody detect CPA4 in mouse or rat samples?

This antibody has been validated against human CPA4. Cross-reactivity with mouse or rat CPA4 has not been formally validated. Human CPA4 shares approximately 80-85 percent sequence identity with mouse and rat orthologs, so cross-reactivity is possible but not guaranteed. If you are working with rodent samples, we recommend testing the antibody at the standard dilution first on a positive control lysate known to express CPA4, then titrating as needed. Include a CPA4-knockout or knockdown control if available to confirm band specificity.

What is the recommended starting dilution for Western blot with this CPA4 antibody?

Start at 1:1000 dilution in your blocking buffer for Western blot applications. This dilution has been validated with standard chemiluminescent detection systems. If your CPA4 expression level is low or you are working with conditioned media rather than whole-cell lysates, you may need to increase antibody concentration to 1:500. Conversely, if background is high, titrate to 1:2000 or 1:3000. Load 20-40 micrograms of total protein per lane as a starting point and include a recombinant CPA4 positive control if signal is weak.

Can I use this antibody for immunohistochemistry on formalin-fixed tissue?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress. For formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required. Citrate buffer (pH 6.0) heat-induced epitope retrieval is a standard starting point for carboxypeptidase antibodies. Use the antibody at a dilution range of 1:50 to 1:200 depending on your detection system and tissue type. Because CPA4 is a secreted protein, staining may appear in the extracellular space and cytoplasm rather than being strictly intracellular. Include a CPA4-negative tissue as a specificity control.

What tissues or cell lines are good positive controls for CPA4 expression?

CPA4 is broadly expressed across human tissues, with relatively high levels reported in prostate, brain, kidney, and certain tumor types. For Western blot positive controls, prostate cancer cell lines such as PC-3 or DU145 are reasonable starting points, as CPA4 has been implicated in prostate biology. Alternatively, normal human kidney or brain lysates can serve as positive controls. If you are studying a specific tissue context, check public expression databases like the Human Protein Atlas or GEO to identify cell lines or tissues with robust CPA4 mRNA and protein expression.

Is CPA4 intracellular or secreted, and how should I prepare samples?

CPA4 is a secreted metalloprotease. It is synthesized with a signal peptide and released into the extracellular space. For Western blot, you can detect CPA4 in whole-cell lysates, but signal may be stronger in conditioned culture media or serum samples. If analyzing conditioned media, concentrate samples by precipitation or ultrafiltration before loading to improve sensitivity. Use protease inhibitor cocktails during lysis to prevent degradation, and include EDTA-free inhibitors if you plan downstream enzymatic assays, since CPA4 is a zinc-dependent enzyme that may be inhibited by metal chelators.

Are there CPA4 isoforms or splice variants I should be aware of?

The primary CPA4 transcript encodes the 421-amino-acid precursor. Alternative splice variants have been reported in sequence databases, but the functional significance and expression levels of these isoforms are not well characterized. The canonical isoform is most commonly studied and is the expected target of this antibody. If you observe multiple bands on Western blot that do not correspond to post-translational modifications, consider the possibility of splice variants or proteolytic fragments. Sequence the band by mass spectrometry if isoform distinction is critical to your experiment.

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

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied at 100 micrograms and is stable for at least 12 months from date of receipt when stored properly. For short-term use, an aliquot can be kept at 4°C for up to one month. Avoid prolonged exposure to room temperature. If you observe particulates after thawing, centrifuge briefly before use. Adding sodium azide (0.02-0.05 percent final concentration) or glycerol (50 percent) can extend stability for long-term storage at -20°C or 4°C, respectively.