Anti-Calpain-7 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CAPN7 on Western blot?

Calpain-7 migrates at approximately 90-95 kDa on SDS-PAGE, corresponding to the full-length 813 amino acid protein. You may observe additional lower molecular weight bands representing proteolytic fragments or degradation products, particularly in lysates prepared without protease inhibitors. Nuclear extracts typically yield cleaner signals than whole-cell lysates due to CAPN7's predominant nuclear localization. If you see multiple bands, consider running a parallel blot with nuclear and cytoplasmic fractions to confirm the identity of the principal band.

Does this antibody work for mouse and rat Calpain-7 detection?

This antibody has been validated for human CAPN7 and shows predicted cross-reactivity with mouse and rat orthologs based on epitope sequence homology. However, we have not experimentally confirmed reactivity in these species. If working with rodent samples, we recommend starting with the 1:1000 dilution used for human and running a positive control lysate from stomach tissue, where CAPN7 expression is highest across species. Titrate the antibody if signal intensity is suboptimal.

What tissues or cell lines are good positive controls for CAPN7?

Stomach tissue serves as the strongest positive control, as CAPN7 exhibits particularly high tissue-specific expression in gastric epithelium. Among cell lines, those of gastric origin or cells undergoing active cytokinesis provide reliable signal. HeLa cells synchronized in late telophase show robust CAPN7 localization at the midbody. Decidualizing endometrial stromal cells also express elevated CAPN7. Avoid using liver or skeletal muscle as positive controls, as CAPN7 expression is minimal in these tissues compared to classical calpains.

Should I use nuclear extraction for CAPN7 Western blots?

Nuclear extraction is advisable but not strictly required. CAPN7 localizes predominantly to the nucleus, so nuclear fractionation enriches your target and reduces background from abundant cytoplasmic proteins. This approach improves signal-to-noise ratio and simplifies interpretation. If performing whole-cell lysis, use RIPA or a similarly stringent buffer to solubilize nuclear compartments effectively. Include benzonase or another nuclease to reduce viscosity. For immunofluorescence applications, standard methanol or paraformaldehyde fixation provides adequate access to nuclear CAPN7 without specialized permeabilization.

What dilution should I start with for IHC on paraffin sections?

For paraffin-embedded tissue, start with a 1:200 to 1:500 dilution range following antigen retrieval. Citrate buffer (pH 6.0) heat-induced epitope retrieval typically works well for calpain family members. Given CAPN7's nuclear localization, you should observe distinct nuclear staining in positive tissues like gastric mucosa. Optimize dilution on your specific tissue type, as fixation time and processing conditions influence antibody accessibility. For frozen sections, a 1:500 starting dilution is reasonable, though signal may be stronger due to better epitope preservation.

How do I distinguish CAPN7 from other calpain family members on a blot?

CAPN7 runs at approximately 90-95 kDa, which distinguishes it from the more abundant classical calpains (CAPN1 at 80 kDa, CAPN2 at 80 kDa). Its nuclear localization also provides orthogonal confirmation through fractionation experiments. If concerned about cross-reactivity, perform a competition assay with recombinant CAPN7 peptide or include lysates from CAPN7 knockout cells if available. The tissue-specific expression pattern, with strong signal in stomach and weak signal in skeletal muscle, further differentiates CAPN7 from ubiquitously expressed classical calpains.

Can this antibody detect endogenous CAPN7 or only overexpressed protein?

This polyclonal antibody detects endogenous CAPN7 in tissues and cell types where expression is naturally elevated, particularly gastric tissue and cells undergoing cytokinesis. In cell lines with low basal CAPN7 expression, detection may require longer exposures or signal amplification. The recommended 1:1000 dilution for Western blot is optimized for endogenous protein levels in stomach lysates. For less abundant samples, you may need to load 30-50 μg total protein per lane or enrich via nuclear fractionation to achieve reliable detection without resorting to overexpression systems.

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

Store the antibody at -20°C in the original packaging. Avoid repeated freeze-thaw cycles; for frequent use, aliquot upon receipt into single-use volumes. The antibody remains stable for at least 12 months when stored properly at -20°C. For working stocks, diluted antibody in blocking buffer containing 0.02% sodium azide can be stored at 4°C for up to one week and reused for multiple blots. Do not store diluted antibody without preservative for more than a few days, and never refreeze aliquots after thawing.