CASP3 Recombinant Monoclonal Antibody

What is the difference between antibodies targeting pro-CASP3 versus active CASP3?

Pro-CASP3 antibodies recognize the inactive zymogen form (~32 kDa), while active CASP3 antibodies specifically detect the cleaved fragments (primarily p17/p19) generated during apoptosis. Some antibodies can detect both forms. The selection depends on your experimental goals:

• Pro-CASP3 antibodies: Useful for monitoring total CASP3 expression levels regardless of activation status

• Active CASP3 antibodies: Specifically mark cells undergoing apoptosis by detecting the p17 subunit but not precursor forms

• Dual-specificity antibodies: Detect both forms, allowing tracking of CASP3 processing dynamics

For example, the anti-active CASP3 antibody from R&D Systems (AF835) specifically detects the p17 subunit but not the precursor form, making it ideal for distinguishing apoptotic from non-apoptotic cells . Conversely, some antibodies like Boster Bio's M00334-3 can recognize multiple forms (p17, p19, and p32) .

How do monoclonal, polyclonal, and recombinant monoclonal CASP3 antibodies differ in research applications?

Recombinant monoclonal antibodies offer significant advantages for reproducibility. As noted in search results, "recombinant production enables lot-to-lot consistency and is animal-cruelty-free" . These antibodies are produced using in vitro expression systems by cloning specific antibody DNA sequences, resulting in better specificity, sensitivity, and consistency between lots .

What applications are CASP3 recombinant monoclonal antibodies typically validated for?

Most CASP3 recombinant monoclonal antibodies are validated for multiple applications:

For example, Boster Bio's anti-active CASP3 antibody has been validated for IF, IHC, ICC, and WB applications specifically with human samples .

How should I select optimal controls for CASP3 antibody validation in apoptosis studies?

Proper controls are essential for validating CASP3 antibody specificity:

Positive Controls:

• Cell lines treated with known apoptosis inducers:

  • Jurkat cells treated with staurosporine (0.5 μM, 10-18 hours)

  • HeLa cells treated with apoptosis inducers

• Recombinant active CASP3 protein (CC119/human CASP3 incubated with 1 mM ATP for 30 minutes)

Negative Controls:

• Untreated healthy cells

• Cells with CASP3 knockdown or knockout

• Pre-incubation with blocking peptide

According to protocol recommendations: "For neuronal culture preparations, incubation with staurosporine (0.5 μM, 10-18 hr) typically gives a strong caspase 3 signal" . Additionally, some antibodies like HuaBio's ET1608-64 are specifically validated with knockout/knockdown samples for definitive confirmation of specificity .

What factors should I consider when optimizing antigen retrieval for CASP3 IHC?

Antigen retrieval is critical for successful CASP3 IHC:

• Buffer options:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative option)

• Method considerations:

  • Heat-induced epitope retrieval is generally preferred

  • For formalin-fixed paraffin-embedded tissues, more aggressive retrieval may be necessary

• Optimization variables:

  • Temperature and duration

  • Antibody concentration (typically 1:50-1:200 for IHC)

  • Incubation time (generally 1-3 hours at room temperature)

For example, Proteintech's protocol recommends: "suggested antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" .

How can I distinguish between different cleaved forms of CASP3 in Western blots?

Distinguishing between CASP3 forms requires careful experimental design:

• Gel percentage selection: Use 12-15% gels for better separation of the smaller cleaved fragments

• Molecular weight markers:

  • Pro-CASP3: ~32-35 kDa

  • Cleaved p19 fragment: ~19 kDa

  • Cleaved p17 fragment: ~17 kDa

  • Some antibodies may detect heterocomplexes at 50-70 kDa

• Antibody selection: Use antibodies that can recognize multiple forms simultaneously for comparative analysis

• Sample processing: Include both positive controls (apoptotic cells) and negative controls (healthy cells)

The Proteintech antibody data sheet notes: "This antibody can recognize p17, p19 and p32 of Caspase 3" , making it suitable for tracking multiple CASP3 forms simultaneously.

What are the recommended dilutions for different applications of CASP3 antibodies?

As noted in Proteintech documentation: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and may be "sample-dependent" .

How should CASP3 antibodies be stored to maintain optimal activity?

Most CASP3 antibodies require specific storage conditions:

• Long-term storage: -20°C for up to one year

• Short-term/frequent use: 4°C for up to one month

• Avoid: Repeated freeze-thaw cycles

For example, Boster Bio recommends: "Store at -20°C for one year. For short term storage and frequent use, store at 4°C for up to one month. Avoid repeated freeze-thaw cycles" .

Most antibodies are supplied in a storage buffer containing:

• PBS (pH 7.4)

• 0.02% sodium azide

• 50% glycerol

• Sometimes BSA (0.4-0.5 mg/ml)

What technical challenges might arise when detecting low levels of active CASP3 in tissue samples?

Detecting low levels of active CASP3 in tissues presents several challenges:

• Sensitivity limitations:
  "Note when using tissue, levels of active caspase 3 can be below detectable levels"

• Solutions:

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Employ more sensitive detection methods like VisUCyte™ HRP Polymer detection reagents

  • Consider using positive controls to validate detection system

  • Optimize antigen retrieval and antibody concentration

• Validation approaches:

  • Parallel analysis with TUNEL or other apoptosis markers

  • Compare with known apoptotic samples

R&D Systems notes the challenge directly: "levels of active caspase 3 can be below detectable levels" , suggesting the use of appropriate positive controls for validation.

How does epitope selection affect experimental outcomes when using CASP3 antibodies?

Epitope selection is critical when designing CASP3 antibody experiments:

• Conformational vs. Linear Epitopes:

  • Antibodies recognizing linear epitopes may work in Western blot but fail in IHC where protein conformation remains intact

  • Different applications may require antibodies recognizing different epitope types

• Post-translational modifications:

  • If an epitope is near phosphorylation, glycosylation, or acetylation sites, modifications may alter binding

  • Consider whether the epitope might be masked or modified during apoptosis

• Strategic epitope selection:

  • For active CASP3 detection, epitopes in the p17 fragment are optimal

  • For total CASP3, epitopes in conserved regions are preferred

As noted in the PTG Lab blog: "Carefully choosing your antibodies based on epitope binding site can greatly influence experimental outcomes, therapeutic strategies, and diagnostic accuracy" .

How can CASP3 antibodies be used to quantify apoptosis in different experimental systems?

CASP3 antibodies enable multiple approaches to quantify apoptosis:

• Immunofluorescence quantification:

  • Count active CASP3-positive cells as a percentage of total cells

  • Combine with nuclear morphology assessment (DAPI staining for condensed/fragmented nuclei)

• Flow cytometry analysis:

  • Quantify percentage of CASP3-positive cells in a population

  • Can be combined with other apoptotic markers

• Western blot densitometry:

  • Measure the ratio of cleaved to total CASP3

  • Normalize to housekeeping proteins

• Tissue section analysis:

  • Count active CASP3-positive cells per field or tissue area

  • Examples include quantification of apoptotic neuronal death as shown in R&D Systems validation data

The search results provide several examples of quantitative approaches: "Quantification of apoptotic neuronal death (apoptotic nuclear profiles, 'apo'; and active caspase-3 positive neurons, 'casp3') from cultures" .

What are the considerations for using CASP3 antibodies in multiplexed immunofluorescence experiments?

When designing multiplexed immunofluorescence with CASP3 antibodies:

• Antibody compatibility:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential staining protocols

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider signal intensity (CASP3 may require brighter fluorophores if expression is low)

• Validated combinations:

  • CASP3 + Ki-67 (proliferation) + nuclear stain (DAPI)

  • CASP3 + cell-type specific markers

  • CASP3 + other apoptotic pathway components

• Controls:

  • Single-stained controls for spectral unmixing

  • Isotype controls to assess background

Examples in the search results include dual staining: "Double immunofluorescent staining to caspase-3 and DAPI" and "Merging of F + G shows green caspase-3-positive apoptotic nuclei and blue non-apoptotic nuclei" .

How are CASP3 recombinant monoclonal antibodies being used in neurodegeneration research?

CASP3 antibodies have important applications in neurodegeneration research:

• Alzheimer's disease studies:

  • CASP3 is "the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease"

• Parkinson's disease models:

  • Used to study LRRK2-induced neuronal death mechanisms

  • Research shows "genetic deletion of Bax prevents mutant LRRK2 induced neuronal death" as assessed by CASP3 activation

• Experimental approaches:

  • Primary neuronal cultures transfected with disease-associated genes

  • Treatment with neurotoxins followed by CASP3 activation assessment

  • In vivo models with subsequent tissue analysis

The search results describe specific examples: "Primary embryonic cortical neurons are derived from WT mice or mice lacking the pro-apoptotic protein Bax, and transiently transfected with GFP-tagged WT or mutant LRRK2 (R1441C, G2019S)" .

What are the technical considerations for using CASP3 antibodies in cell-based assays versus tissue sections?

Different sample types require distinct approaches:

For cell-based work: "Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X-100/PBS" .

For tissue work: "Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic" .

How can CASP3 antibodies be integrated with other apoptosis detection methods for comprehensive analysis?

A multi-method approach provides stronger evidence of apoptosis:

• Complementary apoptosis detection methods:

  • TUNEL assay (DNA fragmentation)

  • Annexin V staining (phosphatidylserine externalization)

  • Mitochondrial membrane potential assays

  • JC-1 staining

• Integrated analysis approaches:

  • Sequential staining of the same sample

  • Parallel analysis of adjacent sections

  • Correlation of CASP3 activation with other markers

• Validation examples from research:

  • Studies showing CASP3 activation preceding phosphatidylserine exposure

  • CASP3-mediated cleavage of scramblase proteins: "Cleaves phospholipid scramblase proteins XKR4, XKR8 and XKR9, leading to promote phosphatidylserine exposure on apoptotic cell surface"

This integrated approach allows researchers to distinguish between different forms of cell death (apoptosis vs. necrosis vs. pyroptosis) and to establish temporal relationships between different apoptotic events.