Anti-Calpain-8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Calpain-8 on Western blot?

Full-length human CAPN8 migrates at approximately 80 kDa on SDS-PAGE, consistent with its 703-residue sequence. In gastric mucosal lysates you may observe this predominant band along with lower-molecular-weight species around 60-65 kDa representing autolytic or proteolytic fragments, particularly if calcium was present during lysis. To minimize autoproteolysis, include 5 mM EDTA or EGTA in your extraction buffer. Some laboratories report doublets near 80 kDa in stomach tissue, likely reflecting post-translational modifications or alternative translation initiation. Start with 20-40 µg total protein per lane at 1:1000 dilution.

Is this Calpain-8 antibody suitable for mouse and rat gastric tissue?

The antibody is validated against human CAPN8 and shows potential cross-reactivity with mouse and rat orthologs based on sequence homology in the immunogenic region. Mouse Capn8 shares approximately 85% identity with human CAPN8, and rat is similarly conserved. We recommend empirical testing with positive control lysates from mouse or rat stomach mucosa before committing to a full experiment. Start at 1:1000 dilution and include a known human gastric sample on the same blot for comparison. Cross-reactivity has not been formally validated in our hands, so titre the antibody against your specific rodent tissue.

What tissue samples work as positive controls for Calpain-8 experiments?

Human gastric mucosa represents the best positive control, as CAPN8 is most abundantly expressed in surface mucus cells of the stomach. Fresh-frozen or snap-frozen gastric biopsy material yields stronger signal than formalin-fixed paraffin-embedded sections. If primary human tissue is unavailable, gastric cancer cell lines such as AGS or MKN45 express detectable but lower levels of CAPN8. For embryonic studies involving convergent extension, you may detect CAPN8 in Xenopus or zebrafish gastrula-stage lysates, though expression is lower. Avoid liver, brain, or skeletal muscle as these tissues show minimal to undetectable CAPN8 by Western blot.

Should I add calcium or EDTA when preparing lysates for Calpain-8 Western blots?

Add 5 mM EDTA or EGTA to your lysis buffer unless you specifically aim to study calcium-dependent autoproteolysis. CAPN8 is a calcium-activated cysteine protease and will undergo autolytic cleavage if free calcium is present during extraction, generating lower-molecular-weight fragments that complicate interpretation. Standard RIPA buffer supplemented with protease inhibitor cocktail and EDTA preserves full-length CAPN8 effectively. If your experimental design requires calcium treatment, perform it on intact cells before lysis, then immediately quench with excess EDTA and process samples on ice to halt further proteolysis.

Can I use this antibody to distinguish Calpain-8 from other calpain family members?

This polyclonal antibody was raised against a CAPN8-specific epitope and does not cross-react with the classical calpains (CAPN1, CAPN2) or other tissue-specific isoforms in our Western blot validation panel. The catalytic domains of calpain family members share considerable homology, but CAPN8 contains unique N-terminal and domain-III sequences. If you work with samples co-expressing multiple calpains, verify specificity by running lysates from tissues where CAPN8 is absent (e.g., liver for CAPN1/2 alone) alongside gastric samples. Immunoprecipitation followed by mass spectrometry can confirm identity in complex samples.

What starting dilution should I use for immunohistochemistry on gastric sections?

Begin at 1:200 to 1:500 for paraffin-embedded human gastric tissue sections. Antigen retrieval is essential: heat-induced epitope retrieval in citrate buffer, pH 6.0, for 20 minutes yields consistent results. CAPN8 localizes predominantly to the cytoplasm of surface mucus cells in the gastric pits, with minimal signal in chief or parietal cells deeper in the glands. Frozen sections often give stronger signal and can tolerate dilutions up to 1:800. Include a known CAPN8-negative tissue (colon or liver) as a negative anatomical control on the same slide when possible.

How should I store the Calpain-8 antibody for long-term stability?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase background. The polyclonal serum remains stable for at least two years under these conditions based on our internal testing. If you use the antibody frequently, a working aliquot can be held at 4°C for up to one month with 0.02% sodium azide as preservative. Do not store diluted antibody in blocking buffer for more than one week. Avoid frost-free freezers, as temperature cycling degrades immunoglobulin over time.

Does Calpain-8 require calcium in the sample for antibody recognition?

No. This antibody recognizes CAPN8 in both calcium-bound and apo (calcium-free) conformations, and in fact we recommend preparing lysates with EDTA to preserve the full-length protease. The epitope recognized by the antibody is not within the calcium-binding EF-hand domains and does not depend on conformational changes induced by calcium binding. You will detect CAPN8 equally well in resting cells and in calcium-stimulated cells, though the banding pattern may differ due to autoproteolytic processing in the latter. Denaturation during SDS-PAGE eliminates any calcium-dependent structure, so Western blot detection is conformation-independent.