Anti-Calpain Small Subunit 2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CAPNS2 on Western blot?

CAPNS2 migrates at approximately 28-30 kDa on Western blot, corresponding to the 248-amino acid full-length protein. You may observe a doublet or slight molecular weight variation depending on post-translational modifications or proteolytic processing. The small subunit remains relatively stable compared to the catalytic large subunits, so you should see a single predominant band in most lysates. If you observe higher molecular weight species, consider that CAPNS2 forms stable heterodimers with large calpain subunits, though under reducing SDS-PAGE conditions these complexes typically dissociate.

What is the optimal starting dilution for Western blot with this Calpain-S2 antibody?

Begin with a 1:1000 dilution in blocking buffer containing 5% non-fat milk or BSA in TBST. This rabbit polyclonal typically produces clean signal at this dilution with 20-50 µg total protein per lane. If your signal is weak, particularly in samples with low CAPNS2 expression, you can increase antibody concentration to 1:500. Overnight incubation at 4°C often improves sensitivity compared to one-hour room temperature incubations. Always include a lysate known to express CAPNS2 as a positive control to confirm antibody performance in your hands.

Does this antibody distinguish between CAPNS1 and CAPNS2 isoforms?

The two small subunit isoforms, CAPNS1 and CAPNS2, share approximately 60% sequence identity and similar molecular weights, making isoform-specific detection challenging. This antibody is raised against CAPNS2, but you should verify specificity for your application through siRNA knockdown or CAPNS2-null cell lines if distinguishing between isoforms is critical to your experiment. CAPNS1 is ubiquitously expressed while CAPNS2 shows more restricted tissue distribution, so tissue context may provide some functional inference. For rigorous isoform discrimination, consider parallel knockdown experiments as biological controls.

Will this antibody work in mouse or rat samples?

This antibody has been validated for human CAPNS2. Cross-reactivity with mouse and rat is predicted based on high sequence homology across mammalian species—human and mouse CAPNS2 share approximately 90% identity at the amino acid level. However, we have not formally validated reactivity in rodent tissues. If you are working with mouse or rat lysates, we recommend testing the 1:1000 dilution initially and including a positive control human lysate on the same blot. Researchers in our experience have reported successful detection in rodent samples, but optimal dilution may require adjustment.

What positive control tissues or cell lines express high levels of CAPNS2?

CAPNS2 is broadly expressed across human tissues, with relatively higher abundance in skeletal muscle, heart, and brain based on transcriptomic data. For a convenient positive control, HeLa or HEK293 lysates typically show detectable CAPNS2 expression at standard loading amounts. If you are studying tissue-specific regulation, consider that CAPNS2 expression may be inducible under certain conditions such as differentiation or calcium flux. Preparing lysates from proliferating cultured cells generally provides consistent baseline expression suitable for validating antibody performance before moving to experimental samples.

Can I use this antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are routinely validated for Western blot applications. This rabbit polyclonal should be compatible with both immunofluorescence and immunohistochemistry, though we have specifically validated Western blot performance for this Calpain-S2 antibody. For IHC or IF, start with a 1:100 to 1:200 dilution and optimize based on your fixation method and tissue type. Antigen retrieval may be necessary for formalin-fixed paraffin-embedded tissues—citrate buffer pH 6.0 is a standard starting point. Expect cytoplasmic staining consistent with CAPNS2 localization.

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

Store the antibody at -20°C in the provided glycerol-containing buffer. Avoid repeated freeze-thaw cycles, which can reduce titer and increase background. For routine use, consider preparing small aliquots immediately upon receipt. The antibody remains stable for at least 12 months when stored properly at -20°C. If you use the antibody frequently, a working aliquot can be kept 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, as carrier proteins can degrade and affect performance.

What sample preparation considerations are important for detecting CAPNS2?

Use standard RIPA or NP-40 lysis buffers with protease inhibitors—CAPNS2 is a cytoplasmic protein and extracts efficiently under non-denaturing conditions. Because the calpain system is calcium-dependent, include EDTA or EGTA in your lysis buffer to prevent artifactual proteolysis during sample preparation. CAPNS2 itself is relatively stable, but active calpains in the lysate can cleave other substrates if calcium is present. Boil samples in SDS loading buffer immediately after quantification. For cultured cells, 20-50 µg total protein per lane is typically sufficient. Fresh lysates generally give better results than samples subjected to multiple freeze-thaw cycles.