CAPN7 Antibody

What is CAPN7 and what are its key biological functions?

CAPN7 (Calpain 7) is a ubiquitously expressed cysteine protease belonging to the Peptidase C2 protein family. It is a 92.7 kDa protein composed of 813 amino acid residues in humans with primary subcellular localization in the nucleus . Recent research has revealed that CAPN7 plays crucial roles in:

• Cytokinetic abscission - the final step of cell division where daughter cells physically separate

• NoCut checkpoint maintenance - which delays abscission in response to mitotic errors

• ESCRT-dependent degradation pathways, potentially involving EGFR through endosomal/MVB processing

• Human endometrial stromal cell migration, invasion, and decidualization

Unlike other calpains, CAPN7 contains tandem microtubule-interacting and trafficking (MIT) domains that enable it to interact specifically with the ESCRT-III subunit IST1. This interaction can activate CAPN7's proteolytic activity, though its physiological substrates remain largely unknown .

What types of CAPN7 antibodies are commercially available for research?

Several types of CAPN7 antibodies are available for research applications:

Most commercial antibodies are raised against fusion proteins of human CAPN7 or specific immunogenic regions . When selecting an antibody, it's important to consider both the intended application and the species reactivity needed for your experimental system.

What is the optimal sample preparation technique for CAPN7 detection in Western blot?

For optimal CAPN7 detection in Western blot, the following sample preparation techniques are recommended:

• Lysis buffer composition:

  • Standard RIPA or NP-40 buffer systems

  • Complete protease inhibitor cocktail (critical to prevent CAPN7 autolysis)

  • Phosphatase inhibitors if examining post-translational modifications

• Sample sources reported to yield good results:

  • Human heart tissue, brain tissue

  • Mouse and rat brain tissue

  • HeLa cells

• Sample handling:

  • Keep samples cold throughout preparation

  • Process quickly to minimize degradation

  • Use freshly prepared samples when possible

• Running conditions:

  • 8-10% polyacrylamide gels provide optimal resolution for the 92-93 kDa CAPN7 protein

  • Include ladder markers that accurately define the 90-100 kDa range

• Antibody concentration ranges:

  • Proteintech 26985-1-AP: 1:1000-1:6000

  • Proteintech 13870-1-AP: 1:500-1:1000

  • PAF256Hu01: 0.01-2μg/mL

When analyzing results, expect a primary band at approximately 92-93 kDa. Additional bands may indicate autolysis, which is enhanced by IST1 binding , or post-translational modifications.

How can I verify CAPN7 antibody specificity in my experimental system?

Comprehensive verification of CAPN7 antibody specificity should employ multiple complementary approaches:

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown: Compare antibody staining in cells treated with CAPN7-specific siRNA versus control siRNA

  • CRISPR/Cas9 knockout: Generate CAPN7 knockout cell lines as negative controls

  • Rescue experiments: The research literature describes successful use of siRNA-resistant CAPN7 constructs expressed in cells depleted of endogenous CAPN7

• Structure-guided mutant controls:

  • V18D or F98D mutants: Disrupt IST1 binding but maintain protein structure

  • C290S mutant: Eliminates proteolytic activity while maintaining structure

  • These mutants provide powerful tools to distinguish between specific signal and background

• Localization pattern verification:

  • Nuclear localization in most cell types

  • Midbody localization during cytokinesis, specifically in a double-ring pattern on either side of the central Flemming body

  • Co-localization with IST1 in approximately 80% of IST1-positive midbodies for wild-type CAPN7, while binding mutants show <5% co-localization

• Multiple antibody comparison:

  • Test different antibodies targeting distinct CAPN7 epitopes

  • Compare staining patterns to confirm consistency across antibodies

This multi-faceted validation approach ensures confident interpretation of experimental results with CAPN7 antibodies.

What are the recommended dilutions for CAPN7 antibodies across different applications?

The following table summarizes recommended dilutions for CAPN7 antibodies across different applications based on manufacturer data:

For optimal results, perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) with your specific samples to determine the best signal-to-noise ratio . As noted by manufacturers, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" as performance can be sample-dependent.

What are the optimal conditions for immunohistochemical detection of CAPN7?

For optimal immunohistochemical detection of CAPN7, follow these tissue-specific preparation and staining protocols:

• Antigen retrieval methods:

  • Primary recommended method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0 at standard temperatures and times

• Validated tissue types:

  • Human stomach tissue (Proteintech 26985-1-AP)

  • Human ovary cancer tissue (Proteintech 13870-1-AP)

  • Human intrahepatic cholangiocarcinoma tissue (Proteintech 13870-1-AP)

• Antibody dilutions:

  • Proteintech antibodies: 1:50-1:500

  • Abbexa antibody: 1:25-1:100

  • Cloud-Clone PAF256Hu01: 5-20μg/mL

• Detection systems:

  • Standard biotin/streptavidin-based systems

  • Polymer-based detection systems for enhanced sensitivity

  • DAB substrate provides optimal visualization

• Controls:

  • Positive control: Include validated tissues (stomach, ovary)

  • Negative control: Omit primary antibody while maintaining all other steps

  • Additional control: Use isotype-matched irrelevant antibody

For each new tissue type, a titration of antibody concentration is recommended to determine optimal staining conditions that maximize specific signal while minimizing background.

How does CAPN7 interact with the ESCRT-III machinery during cytokinesis?

CAPN7 interacts with the ESCRT-III machinery during cytokinesis through a specific molecular mechanism that has been characterized through biochemical and structural studies:

Structural basis of interaction:

• CAPN7 contains tandem microtubule-interacting and trafficking (MIT) domains

• These MIT domains bind simultaneously to two distinct MIT interaction motifs (MIMs) on the ESCRT-III subunit IST1

• Crystal structure analysis has revealed the precise interactions between these domains

• Structure-guided point mutations (V18D in the first MIT domain, F98D in the second MIT domain) disrupt IST1 binding both in vitro and in cells

Functional significance in cytokinesis:

• The ESCRT-III machinery mediates membrane fission during cytokinetic abscission

• IST1 localizes in a double-ring pattern on either side of the central Flemming body within the midbody

• IST1 recruits CAPN7 to midbodies through direct binding

• Wild-type CAPN7 colocalizes with IST1 in ~80% of all IST1-positive midbodies, whereas IST1 non-binding mutants show <5% colocalization

• This interaction is required for efficient abscission, as CAPN7 depletion increases midbody-stage cells from 5% to 10% and multinucleate cells from 6% to 22%

Regulatory aspects:

• IST1 binding can activate CAPN7 autolysis and proteolytic activity

• CAPN7 proteolytic activity is required for efficient abscission, as the catalytically inactive C290S mutant fails to rescue abscission defects despite proper localization

• The interaction also plays a critical role in NoCut checkpoint maintenance

This complex interplay between CAPN7 and ESCRT-III machinery represents a critical regulatory mechanism for ensuring proper timing and completion of cytokinesis.

What is the role of CAPN7 proteolytic activity in the NoCut checkpoint?

CAPN7's proteolytic activity plays a crucial role in the NoCut checkpoint (also called the Abscission checkpoint), which delays final cell separation in response to mitotic errors:

CAPN7 and NoCut checkpoint regulation:

• The NoCut checkpoint delays abscission when mitotic errors are detected

• CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance

• Depletion of CAPN7 reduces the percentage of cells stalled at the midbody stage during checkpoint activation

• In cells with Nup153 depletion (which activates the checkpoint), midbody-stage cells increase to 23%

• Co-depletion of CAPN7 reduces this to only 10%, indicating checkpoint failure

• Wild-type CAPN7 expression restores checkpoint function (20% midbodies)

Requirements for CAPN7 function:

• Both IST1-binding and proteolytic activity are essential:

  • The catalytically inactive CAPN7(C290S) mutant localizes properly to midbodies but fails to rescue NoCut checkpoint function

  • IST1-binding mutants (V18D, F98D) neither localize properly nor rescue checkpoint function

  • This indicates that CAPN7 must be both correctly localized and enzymatically active

NoCut activation contexts:

• CAPN7 is required to sustain NoCut signaling across different activation mechanisms:

  • Nup153 depletion

  • Replication stress induced by aphidicolin (DNA polymerase inhibitor)

  • ICRF-193 treatments

• Both CAPN7 and SPAST (another protein with MIT domains) are required for checkpoint maintenance

The physiological substrates of CAPN7 remain unidentified, but the evidence clearly demonstrates that its proteolytic activity is essential for maintaining the NoCut checkpoint, highlighting its importance in safeguarding genomic integrity during cell division.

What experimental approaches can be used to identify CAPN7 substrates?

Identifying CAPN7's physiological substrates requires comprehensive experimental approaches that leverage both proteomics and functional validation:

Proteomics-based approaches:

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with mass spectrometry:

  • Compare protein profiles from cells with wild-type CAPN7 versus catalytically inactive CAPN7(C290S)

  • Identify proteins with altered abundance or post-translational modifications

• N-terminomics approaches:

  • Specifically identify new N-termini generated by CAPN7 proteolytic cleavage

  • Compare samples from cells expressing wild-type versus C290S mutant CAPN7

• Proximity-dependent labeling:

  • Create CAPN7-BioID or CAPN7-APEX2 fusion proteins

  • Identify proteins in close proximity to CAPN7 during specific cell cycle stages

Biochemical validation approaches:

• In vitro cleavage assays:

  • Express and purify recombinant CAPN7

  • Test candidate substrates identified from proteomics studies

  • The search results mention that "GFP-CAPN7 can perform autolysis, and this activity is enhanced by IST1 binding," providing a foundation for such assays

• Midbody-focused approaches:

  • Isolate midbody fractions using established protocols

  • Compare protein profiles between wild-type and CAPN7-depleted cells

  • Focus on proteins that change during abscission timing

Functional validation approaches:

• Mutational analysis:

  • Generate non-cleavable mutants of candidate substrates

  • Test whether these mutations affect abscission or NoCut checkpoint function

  • Correlate with CAPN7 activation timing

• Cell cycle-specific analysis:

  • Synchronize cells at different stages of cytokinesis

  • The research literature describes "thymidine treatment/washout was used to synchronize cell cycles and increase the proportion of midbody-stage cells"

  • Monitor substrate cleavage patterns during abscission versus checkpoint activation

• Context-specific approaches:

  • Compare substrate profiles under normal conditions versus NoCut activation

  • Use the V18D, F98D (IST1-binding deficient) and C290S (catalytically inactive) CAPN7 mutants as controls

Since "physiological substrates have not yet been identified" for CAPN7 , these approaches represent promising strategies to identify the key targets through which CAPN7 regulates abscission timing and completion.

Why might I see multiple bands when using CAPN7 antibodies in Western blot?

Multiple bands when using CAPN7 antibodies in Western blot can arise from several biological and technical factors:

The expected molecular weight for human CAPN7 is approximately 92-93 kDa . Any additional bands should be carefully validated to determine their identity and significance. The catalytically inactive C290S mutant serves as a particularly useful control, as it would eliminate bands resulting from autolysis while maintaining the full-length protein band.

How can I improve weak CAPN7 signal in immunofluorescence experiments?

To enhance weak CAPN7 signal in immunofluorescence experiments, consider these optimization strategies:

• Fixation and permeabilization optimization:

  • Test different fixation methods (4% PFA, methanol, or combined protocols)

  • Optimize permeabilization with different detergents (0.1-0.5% Triton X-100, saponin, or digitonin)

  • Adjust permeabilization time to balance epitope accessibility with structural integrity

• Antigen retrieval enhancement:

  • Implement heat-induced epitope retrieval with TE buffer pH 9.0 (primary recommended method)

  • Try citrate buffer pH 6.0 as an alternative

  • Carefully control heating time and temperature

• Signal amplification techniques:

  • Implement tyramide signal amplification (TSA)

  • Use higher sensitivity fluorophores (e.g., Alexa Fluor 647)

  • Consider quantum dots for brighter, more photostable signals

• Cell cycle considerations:

  • CAPN7 localizes to midbodies specifically during cytokinesis

  • Synchronize cells to enrich for midbody-stage cells

  • Research protocols use "thymidine treatment/washout to synchronize cell cycles and increase the proportion of midbody-stage cells"

• Co-staining strategy:

  • Use IST1 as a co-localization marker

  • Include midbody markers (α-tubulin, Aurora B) to help identify relevant structures

  • The literature reports CAPN7 localizing "in a double-ring pattern on either side of the central Flemming body"

• Antibody optimization:

  • Extend primary antibody incubation to overnight at 4°C

  • Try different antibody concentrations (5-20 μg/mL range recommended for ICC)

  • Consider using mCherry-tagged CAPN7 constructs as mentioned in the research literature

When studying CAPN7 at the midbody, it's particularly important to note that wild-type CAPN7 colocalizes with IST1 in approximately 80% of IST1-positive midbodies, providing a benchmark for successful detection .

What are the most critical considerations when designing experiments with CAPN7 antibodies?

When designing experiments with CAPN7 antibodies, researchers should prioritize these critical considerations:

• Functional context awareness:

  • CAPN7 has dual roles in promoting abscission and maintaining the NoCut checkpoint

  • Consider cell cycle stage when interpreting results, as CAPN7 localization and function change during cytokinesis

  • Include appropriate controls to distinguish between these functions

• Validation strategy:

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to verify antibody specificity

  • Employ the characterized CAPN7 mutants (V18D, F98D, C290S) as powerful specificity controls

  • Validate antibody performance in your specific experimental system before conducting major studies

• Application-specific optimization:

  • Tailor antibody dilutions to specific applications (WB: 1:500-1:6000, IHC: 1:25-1:500, IF: 5-20μg/mL)

  • For difficult applications, consider testing multiple antibodies targeting different CAPN7 epitopes

  • For functional studies, complement antibody-based detection with tagged CAPN7 constructs

• Proteolytic activity considerations:

  • Include protease inhibitors in all buffers to prevent CAPN7 autolysis

  • Be aware that IST1 binding enhances CAPN7 proteolytic activity

  • Consider that both localization and proteolytic activity are required for CAPN7 function

• Physiological relevance:

  • Connect antibody-based observations to functional outcomes

  • Consider that CAPN7's physiological substrates remain unidentified

  • Design experiments that can link CAPN7 localization or activity to cellular processes