ICAD Mouse mAb: Applications, Specificity & Protocol

ICAD Mouse mAb is a highly specific monoclonal antibody targeting ICAD (Inhibitor of Caspase-Activated DNase), also known as DFF45, providing researchers with a reliable tool for investigating apoptotic DNA fragmentation pathways.

Product SKU

Ab351249

Antibody Type

Mouse Monoclonal Antibody (mAb)

Formulation

PBS (Phosphate-Buffered Saline), no carrier proteins or preservatives indicated

Target Antigen

ICAD (Inhibitor of Caspase-Activated DNase) / DFF45

Research Area

Apoptosis, DNA fragmentation, Cell death signaling

Supplier Reference

absea#KC-1231

Background: ICAD and Its Role in Apoptosis

ICAD, or Inhibitor of Caspase-Activated DNase (also designated DFF45 in humans), is a critical regulatory protein in the intrinsic apoptotic pathway. Under normal physiological conditions, ICAD functions as a chaperone and inhibitor for CAD (Caspase-Activated DNase, also known as DFF40), maintaining CAD in an inactive cytoplasmic complex.

During apoptosis, activated caspase-3 cleaves ICAD at specific aspartate residues, releasing the active CAD nuclease. Liberated CAD then translocates to the nucleus, where it cleaves chromosomal DNA at internucleosomal linker regions, generating the ~180 bp oligonucleosomal DNA ladder that is a hallmark of apoptotic cell death. Detection and quantification of ICAD cleavage status therefore serves as a direct molecular readout for caspase-3 activation and commitment to apoptotic DNA fragmentation.

Research on ICAD has implications across cancer biology, neuroscience, immunology, and drug discovery, where modulation of apoptotic pathways is of therapeutic interest.

Product Specifications and Formulation

Aladdin Scientific's ICAD Mouse mAb (SKU: Ab351249) is a mouse-derived monoclonal antibody specifically raised against the ICAD/DFF45 protein. The monoclonal format ensures lot-to-lot consistency and high epitope specificity, which is essential for reproducible detection in complex biological samples.

• Format: Mouse Monoclonal Antibody (IgG subclass to be confirmed with datasheet)
• Formulation: Supplied in PBS (Phosphate-Buffered Saline) only — no glycerol, BSA, or azide preservatives are indicated in the base formulation, making it compatible with a wider range of downstream conjugation or functional assays
• Supplier Reference: absea#KC-1231
• SKU: Ab351249

Researchers should consult the full product datasheet for confirmed details on host species, isotype, immunogen sequence, storage conditions, and concentration prior to experimental use.

Recommended Applications and Usage Guidelines

ICAD Mouse mAb is designed for use in standard immunological assays where detection of ICAD/DFF45 protein expression, abundance, or cleavage status is required. Typical applications for antibodies of this class include:

• Western Blotting (WB): Detection of full-length ICAD (~45 kDa) and/or its caspase-3 cleavage fragments to assess apoptotic commitment in cell or tissue lysates.
• Immunoprecipitation (IP): Isolation of the ICAD–CAD complex or ICAD alone for downstream co-precipitation or activity studies.
• Immunofluorescence (IF) / Immunocytochemistry (ICC): Visualization of ICAD subcellular localization before and after apoptotic stimulus.
• Enzyme-Linked Immunosorbent Assay (ELISA): Quantitative measurement of ICAD levels in cell extracts.

Note: Specific validated applications and working dilutions should be confirmed from the official Ab351249 product datasheet. The above list represents common uses for ICAD monoclonal antibodies and should be treated as guidance pending datasheet confirmation.

General Experimental Protocol for Western Blotting

The following is a general guideline for detecting ICAD using ICAD Mouse mAb by Western blot. Always validate conditions empirically using your specific cell or tissue model.

1. Sample Preparation: Lyse cells in an appropriate lysis buffer (e.g., RIPA buffer supplemented with protease inhibitors). For apoptosis studies, avoid lysis buffers that may further activate caspases ex vivo. Determine protein concentration by BCA or Bradford assay.
2. Gel Electrophoresis: Separate 20–40 µg of total protein per lane on a 10–12% SDS-PAGE gel under denaturing, reducing conditions. ICAD migrates at approximately 45 kDa; its caspase-3 cleavage products appear at lower molecular weights.
3. Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard wet or semi-dry transfer protocols.
4. Blocking: Block membrane for 1 hour at room temperature in 5% non-fat dry milk or 5% BSA in TBST.
5. Primary Antibody Incubation: Incubate with ICAD Mouse mAb (Ab351249) at the manufacturer-recommended dilution (refer to datasheet) in blocking buffer overnight at 4°C.
6. Secondary Antibody: Wash membrane 3× with TBST, then incubate with an appropriate HRP-conjugated anti-mouse IgG secondary antibody for 1 hour at room temperature.
7. Detection: Wash 3× with TBST and detect signal using an enhanced chemiluminescence (ECL) substrate. Image using film or a digital imaging system.

Always include a positive control (cell line known to express ICAD) and a loading control (e.g., β-actin or GAPDH).

Storage, Handling, and Quality Considerations

Proper storage and handling of ICAD Mouse mAb is essential to maintain antibody activity and ensure reproducible experimental results.

• Storage Temperature: Refer to the product datasheet for recommended storage temperature (typically –20°C for long-term storage of mAbs in PBS formulation without glycerol).
• Avoid Freeze-Thaw Cycles: Repeated freeze-thaw cycles can denature antibody protein and reduce binding activity. Aliquot upon receipt for single-use applications.
• PBS-Only Formulation: Since this antibody is supplied in PBS without glycerol or azide, additional precautions for microbial contamination should be taken if storing at 4°C for extended periods. Consider filter sterilization or short-term use at 4°C.
• Quality Control: Each lot of Ab351249 should be accompanied by QC data confirming reactivity and specificity. Request a Certificate of Analysis (CoA) from Aladdin Scientific to verify lot-specific performance data before use.

For technical support or ordering inquiries regarding ICAD Mouse mAb (Ab351249), contact Aladdin Scientific's customer service team.

Frequently asked questions

What is ICAD and why is it an important research target?

ICAD (Inhibitor of Caspase-Activated DNase), also known as DFF45, is a protein that normally inhibits CAD nuclease activity. During apoptosis, caspase-3 cleaves ICAD, releasing active CAD, which then fragments chromosomal DNA — a hallmark event of programmed cell death. It is a key target for apoptosis research, cancer biology, and drug discovery.

What formulation is ICAD Mouse mAb (Ab351249) supplied in?

ICAD Mouse mAb (Ab351249) is supplied in PBS (Phosphate-Buffered Saline) only, with no glycerol, BSA, or azide preservatives indicated in the base formulation. Researchers should consult the product datasheet for full formulation details and storage recommendations.

Which experimental applications is ICAD Mouse mAb suitable for?

ICAD Mouse mAb is commonly used in applications such as Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and ELISA for detection of ICAD/DFF45. Validated applications and recommended working dilutions should be confirmed from the official Ab351249 product datasheet.

How should I confirm the specificity and lot performance of Ab351249 before use?

Request a Certificate of Analysis (CoA) from Aladdin Scientific for the specific lot of Ab351249 you have received. The CoA will include QC data such as validated reactivity, concentration, and tested applications. Always run appropriate positive and negative controls in your experiments.

Why is it important to avoid repeated freeze-thaw cycles with this antibody?

Repeated freeze-thaw cycles can cause protein denaturation, aggregation, and loss of antibody binding activity, leading to reduced assay performance and irreproducible results. Since Ab351249 is formulated in PBS without stabilizing agents like glycerol, aliquoting the antibody upon first receipt is strongly recommended.

References

1. Enari M, et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998.
2. Liu X, et al. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997.
3. Aladdin Scientific. ICAD Mouse mAb (Ab351249) Product Datasheet. Aladdin Scientific.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.