Anti-Calpain-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Calpain-1 on Western blot?

The Calpain-1 catalytic subunit (CAPN1) migrates at approximately 80 kDa on SDS-PAGE, corresponding to its 714-residue sequence. You may observe a doublet or additional lower molecular weight bands representing autolytic cleavage products, particularly if samples were not processed rapidly or contain residual calcium. Calpain-1 undergoes autoproteolysis upon activation, generating fragments around 76 kDa and 18 kDa. To minimize artifactual proteolysis, lyse cells in the presence of EDTA or EGTA and protease inhibitor cocktails, then process samples immediately or snap-freeze in liquid nitrogen.

How do I distinguish Calpain-1 from Calpain-2 in my Western blot?

Calpain-1 (CAPN1, 80 kDa) and Calpain-2 (CAPN2, also 80 kDa) are highly homologous and migrate at nearly identical molecular weights, making separation by size alone unreliable. This antibody is raised against human Calpain-1 and has not been validated for Calpain-2 cross-reactivity. To confirm isoform identity, consider including tissues or cell lines with known differential expression: skeletal muscle and erythrocytes are enriched in Calpain-1, while Calpain-2 predominates in brain and certain epithelial tissues. Alternatively, siRNA knockdown of CAPN1 provides a definitive negative control for antibody specificity.

What is the recommended starting dilution for Calpain-1 Western blot?

Start at 1:1000 dilution in blocking buffer (5% non-fat milk or 3% BSA in TRIS-buffered saline with 0.1% Tween-20) for overnight incubation at 4°C. This rabbit polyclonal typically produces clean signal at this dilution with standard chemiluminescent detection. If background is high, titrate to 1:2000 or 1:5000. For low-abundance samples or brief exposures, 1:500 may improve sensitivity. Calpain-1 is moderately abundant in most adherent cell lines; 20-30 µg total lysate per lane should suffice. Always include a positive control lysate from proliferating cells where Calpain-1 expression is constitutive.

Does this antibody work on mouse or rat samples?

This antibody is validated for human Calpain-1. The catalytic subunit shares approximately 90% sequence identity between human, mouse, and rat, suggesting likely cross-reactivity, but we have not formally validated reactivity in rodent samples. If working with mouse or rat lysates, expect similar banding patterns at 80 kDa but verify specificity with a positive control from the species of interest. Researchers have reported successful detection in mouse tissue lysates at comparable dilutions, but you should titrate and confirm band identity with appropriate knockout or knockdown controls before relying on the data.

Can I use this Calpain-1 antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are routinely validated for Western blot and immunohistochemistry applications. While we have not formally tested this particular Calpain-1 antibody in immunofluorescence in-house, rabbit polyclonals raised against full-length or large domain fragments often perform well in IF protocols. For IHC on paraffin-embedded tissue, antigen retrieval with citrate buffer (pH 6.0) at sub-boiling temperature for 20 minutes is a reasonable starting point. Calpain-1 localizes primarily to the cytoplasm in resting cells and may redistribute to membranes or blebs during calcium signaling or apoptosis. Optimize blocking and dilution empirically, beginning at 1:200 for IHC.

Why am I seeing multiple bands on my Calpain-1 Western blot?

Multiple bands below 80 kDa likely reflect autoproteolytic cleavage. Calpain-1 is a calcium-activated protease that cleaves itself and other substrates when calcium concentrations reach micromolar levels during lysis or freeze-thaw cycles. To reduce artifactual cleavage, supplement lysis buffer with 5 mM EDTA or EGTA, add a cysteine protease inhibitor such as E-64 or leupeptin, and keep samples on ice throughout processing. A band at approximately 76 kDa represents the autolyzed catalytic subunit after removal of the N-terminal domain. If bands persist despite rigorous protease inhibition, they may represent endogenous processing in signaling-active or apoptotic cells.

How should I store the Calpain-1 antibody and what is the expected 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 100 µg format should be aliquoted upon receipt into 10-20 µL volumes sufficient for one or two experiments. Polyclonal antibodies in glycerol-containing storage buffer remain stable for at least 12 months at -20°C under these conditions. For frequent use, a working aliquot may be held at 4°C for up to one month, but do not store diluted antibody in blocking buffer for more than one week. Avoid repeated freeze-thaw of the stock vial; each cycle can decrease functional titer by 10-20 percent.

What positive and negative controls should I include when using this Calpain-1 antibody?

HeLa, HEK293, or Jurkat cell lysates serve as reliable positive controls, as Calpain-1 is constitutively expressed in proliferating mammalian cells. For negative controls, consider lysate from cells transfected with CAPN1-targeting siRNA, though incomplete knockdown is common due to protein stability. Erythrocyte ghosts, which are enriched in Calpain-1, provide an additional positive control with minimal Calpain-2 contamination. As a procedural control, run one lane with secondary antibody alone to assess non-specific binding. Because Calpain-1 can be cleaved during sample handling, include a freshly prepared lysate alongside frozen samples to assess processing artifacts.