CAPN5 Antibody

What is CAPN5 and what cellular functions does it perform?

CAPN5 (Calpain-5) is a calcium-dependent cysteine protease involved in proteolysis and signal transduction pathways. In humans, the canonical protein consists of 640 amino acid residues with a molecular mass of approximately 73.2 kDa . As a member of the Peptidase C2 protein family, CAPN5 differs from classical calpains (CAPN1 and CAPN2) by containing a C2-like domain instead of a penta-EF hand domain at its C-terminus .

Functionally, CAPN5:

• Participates in calcium-regulated non-lysosomal proteolysis

• Interacts with cytoskeletal proteins

• Promotes degradation or remodeling that facilitates normal cellular function

• Contributes to cellular adaptation during stress responses

• Associates with promyelocytic leukemia protein bodies (PML) in the nucleus, which are implicated in cellular stress response, apoptosis, cellular senescence, and protein degradation

How is CAPN5 expressed across different tissues and species?

CAPN5 demonstrates a varied expression pattern across tissues and species:

In humans:

• Expressed in numerous tissues throughout the body

• Represents the second most abundantly expressed calpain in the central nervous system

• In retinal tissue, specifically localizes to:

  • Outer plexiform layer (OPL)

  • Outer nuclear layer (ONL)

  • Inner and outer segments of photoreceptors

  • Synapses of rod and cone photoreceptors

  • Some ganglion cells

  • Inner plexiform layer

Across species:
CAPN5 orthologs have been identified in multiple species including:

• Mouse

• Rat

• Bovine

• Frog

• Zebrafish

• Chimpanzee

• Chicken

The zebrafish has two CAPN5 orthologs (capn5a and capn5b) with capn5a showing 68% identity and 81% similarity to human CAPN5, while capn5b shows 71% identity and 83% similarity .

What diseases are associated with CAPN5 mutations and what are their mechanisms?

The primary disease associated with CAPN5 mutations is Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV), a devastating inherited autoimmune disease of the eye (OMIM #193235) . ADNIV presents as a bilateral panuveitis and displays features commonly seen in other eye diseases, including:

• Retinitis pigmentosa

• Diabetic retinopathy

• Vitelliform macular dystrophy

• Cone-rod dystrophy

Disease mechanism:
ADNIV results from gain-of-function mutations in CAPN5 that lower the calcium threshold required for activation, resulting in hyperactivity of the protease . Key mutations include:

These mutations are located within the calcium-sensitive domain 2 near the active site and are thought to cause the mislocalization of CAPN5 from the cell membrane to the cytosol .

How has loss-of-function research advanced our understanding of CAPN5 therapeutic approaches?

Loss-of-function (LOF) research has provided critical insights for potential therapeutic approaches targeting CAPN5:

Key findings from genetic inactivation studies:

• Photoreceptor-specific knockout (KO) mice for Capn5 showed no significant retinal abnormalities when examined by histology and electroretinography

• Genetic databases from 60,706 unrelated subjects without severe disease phenotypes revealed 22 LOF CAPN5 variants located throughout the gene and in all major protein domains

• LOF variants were found near known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs

Therapeutic implications:
These findings support that:

• Localized inhibition of CAPN5 is a viable strategy for treating hyperactivating disease alleles

• Small molecule pharmacological inhibition of dominant CAPN5 mutations represents a feasible treatment approach that doesn't require development of individual, patient-specific gene therapy vectors

• The lack of adverse effects from CAPN5 LOF suggests that CAPN5 inhibition, even below normal biological activity levels, could be an efficacious strategy for future clinical trials

What criteria should guide selection of CAPN5 antibodies for specific experimental applications?

When selecting CAPN5 antibodies for research, consider these critical factors:

Application compatibility:

Epitope recognition:

• For full-length protein detection: antibodies targeting conserved domains

• For specific isoform detection: antibodies targeting unique regions

• Common immunogens include:

  • N-terminal region (aa 1-50)

  • Middle region

  • C-terminal region

  • Recombinant fragments (e.g., aa 250-600)

Species reactivity:
Verify cross-reactivity with your experimental species. Many commercially available antibodies react with:

• Human CAPN5

• Mouse CAPN5

• Rat CAPN5

Clone selection:
For monoclonal antibodies, specific clones have been validated for particular applications:

• Clone OTI7D2 for immunohistochemistry on paraffin-embedded tissues

• Clone OTI4H5 for Western blot applications

• Clone N1C1 for Western blot, ICC, and IF applications

What validation experiments are essential before using CAPN5 antibodies in advanced research?

Before employing CAPN5 antibodies in complex experiments, comprehensive validation is critical:

1. Specificity validation:

• Western blot analysis comparing wild-type samples with:

  • CAPN5 knockout/knockdown models

  • Overexpression systems (e.g., HEK293T cells transfected with pCMV6-ENTRY CAPN5 cDNA)

• Peptide competition assays to confirm epitope specificity

• Cross-reactivity assessment if working with multiple species

2. Sensitivity assessment:

• Determine lower detection limits using serial dilutions of recombinant CAPN5 protein

• Test antibody performance on endogenous CAPN5 across different tissue types, especially those with varying expression levels (brain tissue shows high expression)

3. Reproducibility testing:

• Perform replicate experiments under identical conditions

• Test lot-to-lot consistency if using multiple antibody batches

4. Application-specific validation:

• For IHC: Include proper controls (e.g., CAPN5-positive tissues like colon and prostate carcinoma)

• For IF: Validate subcellular localization patterns (e.g., photoreceptor inner segments and synaptic terminals in retinal sections)

• For WB: Confirm expected molecular weight (73kDa canonical form, though observed range can be 65-73kDa)

5. Functional validation:

• Compare antibody detection with gene expression data

• Verify antibody recognition of mutant CAPN5 forms if studying disease variants

How should CAPN5 antibodies be optimized for immunohistochemical studies of retinal tissues?

Optimizing CAPN5 immunohistochemistry for retinal tissue requires special considerations:

Tissue preparation protocol:

• Fix retinal tissues in 4% paraformaldehyde for 24 hours

• Paraffin-embed tissues following standard protocols

• Section tissues at 5-7μm thickness

• For antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow sections to cool at room temperature for 20 minutes

Antibody protocol optimization:

• Block non-specific binding with 10% normal serum (matched to secondary antibody host) in PBS with 0.3% Triton X-100

• Incubate with primary anti-CAPN5 antibody:

  • Start with 1:150 dilution as a baseline (for monoclonal antibodies like OTI7D2)

  • For polyclonal antibodies, a wider range (1:50-1:200) may be tested

  • Incubate overnight at 4°C

• Wash 3× in PBS

• Apply appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Develop using DAB and counterstain with hematoxylin

Special considerations for retinal tissues:

• Retinal autofluorescence can interfere with immunofluorescence detection; Sudan Black B (0.1% in 70% ethanol) treatment for 5 minutes after secondary antibody incubation can reduce autofluorescence

• For dual labeling with retinal cell markers, use:

  • Rhodopsin for rod photoreceptors

  • Cone arrestin for cone photoreceptors

  • Glutamine synthetase for Müller glia

  • RBPMS for retinal ganglion cells

Expected localization patterns:
In normal retina, CAPN5 should localize to:

• Photoreceptor inner segments

• Outer plexiform layer (photoreceptor synaptic terminals)

• Some ganglion cells

• Inner plexiform layer

What are the best practices for using CAPN5 antibodies in Western blot applications?

For optimal Western blot detection of CAPN5:

Sample preparation:

• Extract proteins using RIPA buffer supplemented with:

  • Protease inhibitor cocktail

  • 1mM PMSF

  • 1mM sodium orthovanadate

  • 1mM sodium fluoride

• Determine protein concentration using Bradford or BCA assay

• Load 5-30μg of total protein per lane (tissue-dependent)

Protocol optimization:

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal separation

  • Include positive control (e.g., brain tissue lysate)

  • Consider including HEK293T cells transfected with CAPN5 expression vector as additional positive control

• Transfer conditions:

  • Use PVDF membrane (0.45μm pore size)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary antibody in 5% BSA in TBST:

    • 1:500-1:2000 for polyclonal antibodies

    • Follow manufacturer's recommendations for monoclonal antibodies

  • Incubate overnight at 4°C with gentle rocking

• Detection:

  • Use HRP-conjugated secondary antibody (1:5000-1:10000)

  • Develop using enhanced chemiluminescence (ECL)

Expected results:

• Canonical CAPN5 band at approximately 73 kDa

• Observed molecular weight can range from 65-73 kDa

• May detect additional bands due to post-translational modifications or proteolytic processing

Troubleshooting guidance:

• High background: Increase blocking time or use different blocking agent (BSA instead of milk)

• No signal: Verify lysate quality, increase protein loading, or reduce antibody dilution

• Multiple bands: Validate specificity with knockout/knockdown controls or peptide competition

How can CAPN5 antibodies be utilized in studying retinal disease progression and therapies?

CAPN5 antibodies provide valuable tools for retinal disease research:

Disease progression monitoring:

• Comparative immunohistochemistry across disease stages:

  • Use multimodal imaging to correlate CAPN5 expression with clinical findings

  • Compare CAPN5 distribution in normal vs. ADNIV retinal tissues

  • Track changes in expression levels and localization patterns during disease progression

• Proteomics applications:

  • Employ CAPN5 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify CAPN5 substrates and interaction partners in healthy vs. diseased states

  • Shotgun proteomic analysis has revealed 216 differentially-expressed proteins between CAPN5-NIV and control vitreous

Therapeutic development:

• Target validation:

  • Use antibodies to verify knockdown efficacy in siRNA/shRNA approaches

  • Confirm CRISPR-mediated gene editing outcomes

  • Monitor CAPN5 expression in gene therapy models

• Pharmacodynamic marker:

  • Apply CAPN5 antibodies to assess impact of small molecule inhibitors on:

    • Protein expression levels

    • Subcellular localization

    • Protein-protein interactions

• Regenerative medicine applications:

  • Track CAPN5 expression during retinal regeneration in zebrafish models

  • Monitor expression in Müller glia following acute light exposure, suggesting a role in the regenerative process

Clinical translation potential:

• CAPN5 antibodies can help evaluate the efficacy of localized CAPN5 inhibition strategies

• Enable monitoring of treatment response in preclinical models prior to human trials

What approaches enable investigation of CAPN5 activity regulation in complex cellular systems?

Investigating CAPN5 regulation requires sophisticated experimental approaches:

Calcium-dependent activation studies:

• Use calcium imaging techniques (e.g., Fura-2 AM) in combination with immunofluorescence to correlate calcium fluctuations with CAPN5 activation

• Employ calpain activity assays alongside CAPN5 immunoprecipitation to distinguish CAPN5 activity from other calpains

• Examine structural changes using:

  • Fluorescence resonance energy transfer (FRET) sensors

  • Conformational antibodies that recognize active vs. inactive CAPN5 forms

Post-translational modification analysis:

• Combine CAPN5 immunoprecipitation with:

  • Phospho-specific antibodies

  • Ubiquitin detection antibodies

  • Mass spectrometry to identify specific modification sites

• Study how disease-causing mutations affect:

  • The proteolytic core structure

  • Calcium binding capacity

  • Activation thresholds

Subcellular localization dynamics:

• Use confocal microscopy with CAPN5 antibodies alongside organelle markers to track:

  • Nuclear-cytoplasmic shuttling

  • Association with promyelocytic leukemia protein (PML) bodies

  • Membrane association vs. cytosolic distribution

• Employ live-cell imaging with GFP-tagged CAPN5 validated against antibody staining patterns

Genetic approaches for regulatory mechanism studies:

• CRISPR-Cas9 editing to:

  • Introduce disease-specific mutations (p.Arg243Leu, p.Leu244Pro, etc.)

  • Create reporter constructs at endogenous loci

• Conditional knockout/knockin systems to study:

  • Developmental timing of CAPN5 function

  • Tissue-specific regulation

  • Age-dependent changes in expression and function

Translational research applications:

• Patient-derived cells (e.g., iPSCs differentiated to retinal organoids) immunostained for CAPN5

• Zebrafish models with fluorescent reporters validated against antibody staining to visualize real-time regulation in vivo

How might emerging proteomic approaches enhance CAPN5 antibody applications in studying inflammatory pathways?

Advanced proteomic methodologies offer new opportunities for CAPN5 research:

Proximity-dependent labeling techniques:

• BioID or APEX2 fusion with CAPN5 to identify proximal proteins in:

  • Normal vs. disease states

  • Different subcellular compartments

  • Response to calcium fluctuation

• Validate protein interactions using CAPN5 antibodies for co-immunoprecipitation and immunofluorescence co-localization

Temporal proteomic approaches:

• Pulse-chase experiments with CAPN5 antibody pulldown to track:

  • CAPN5 substrate degradation kinetics

  • Protein complex formation and disassociation

• Time-course analysis of proteomic changes during:

  • Acute inflammatory responses

  • Progressive stages of ADNIV pathology

  • Treatment with anti-inflammatory agents

Single-cell proteomics integration:

• Combine CAPN5 antibody-based immunohistochemistry with:

  • Imaging mass cytometry for multiplexed protein detection

  • Digital spatial profiling to correlate CAPN5 with inflammatory markers

• Integrate findings with single-cell transcriptomics data to create comprehensive models of CAPN5 regulation

Vitreous proteome analysis:
The vitreous proteome of CAPN5-NIV patients has revealed pathway alterations including:

• Inflammatory mediators of the acute phase response

• Complement cascade activation

• Decreased synaptic signaling proteins

These pathways share characteristics with:

• Non-infectious posterior uveitis

• Rhegmatogenous retinal detachment (RRD)

• Age-related macular degeneration (AMD)

• Proliferative diabetic retinopathy (PDR)

• Proliferative vitreoretinopathy (PVR)

What role might CAPN5 antibodies play in developing zebrafish models for retinal regeneration research?

Zebrafish models offer unique advantages for CAPN5 research and retinal regeneration studies:

Developmental expression profiling:

• Whole-mount immunohistochemistry with CAPN5 antibodies to:

  • Track expression from embryonic stages through adulthood

  • Map spatial distribution in developing CNS and retina

  • Compare capn5a and capn5b expression patterns and timing

• Key developmental findings:

  • Both capn5a and capn5b are maternally deposited (detectable at 4 hpf)

  • Expression increases gradually throughout development

  • capn5a expression is consistently higher than capn5b (except at 4 hpf)

Photoreceptor-specific functions:

• CAPN5 antibody immunohistochemistry in zebrafish has revealed:

  • Expression in photoreceptor inner segments

  • Localization to outer plexiform layer containing photoreceptor synaptic terminals

  • Expression specific to cone photoreceptors rather than rods (unlike in mammals)

Regeneration research applications:

• CAPN5 antibody staining in injury models has shown:

  • Upregulation in response to photoreceptor degeneration

  • Expression in Müller glia following acute light exposure

  • Potential role in the regenerative process

• Experimental approaches using CAPN5 antibodies:

  • Track expression changes during different phases of regeneration

  • Identify cell types expressing CAPN5 during regenerative response

  • Compare with mammalian models to identify key differences in regenerative capacity

Translational potential:

• Identify pathways where CAPN5 influences regenerative capacity

• Develop drug screening platforms in zebrafish to test CAPN5 modulators

• Create transgenic zebrafish with fluorescent reporters validated against antibody staining to monitor real-time CAPN5 dynamics during regeneration