INCA1 Antibody

What is INCA1 and what is its primary molecular function?

INCA1 functions as a novel cyclin-dependent kinase (CDK) inhibitor that specifically binds to CDK2-bound cyclins and inhibits the kinase activity of CDK2. This binding to cyclins is critical for its function as a CDK inhibitor . On a cellular level, INCA1 inhibits cell growth and proliferation and plays a significant role in cell cycle control. Additionally, it is required for ING5-mediated regulation of S-phase progression, enhancement of Fas-induced apoptosis, and inhibition of cell growth .

INCA1 is encoded on chromosome 17p13 in humans, with chromosome 11B being the mouse homolog location. The human protein consists of 221 amino acids, while the mouse variant contains 231 amino acids .

What are the common commercial INCA1 antibodies and their specifications?

Currently available INCA1 antibodies include:

The immunogen for PA5-60689 is a sequence of "KKRRPCLEGM QQQGLGGVPA RVRAVTYHLE DLRRRQSIIN ELKKAQWGSS GAASEPVVLG EEGCGFPSTN EYPD" , while A49436 was raised against a 15 amino acid peptide from near the carboxy terminus of human INCA1 .

What are recommended handling and storage conditions for INCA1 antibodies?

INCA1 antibodies should be stored at -20°C and remain stable for one year under proper storage conditions. As with all antibodies, repeated freeze-thaw cycles should be avoided to maintain antibody integrity and performance. Antibodies should not be exposed to prolonged high temperatures .

For working solutions, INCA1 antibodies are typically supplied in PBS containing 0.02% sodium azide . When handling these antibodies, standard laboratory safety precautions should be followed, particularly due to the presence of sodium azide, which is toxic.

What are validated applications for INCA1 antibodies in research?

Current commercial INCA1 antibodies have been validated for:

• Western Blot (WB): For detecting INCA1 protein expression levels in cell or tissue lysates

• ELISA (E): For quantitative measurement of INCA1 protein

Western blot validation has been performed using EL4 cell lysate with INCA1 antibody at concentrations of 1 and 2 μg/mL . When designing experiments, researchers should consider these validated applications and cell systems as starting points.

How can I optimize Western blot protocols for INCA1 detection?

For optimal Western blot detection of INCA1:

• Sample preparation: Use fresh lysates from appropriate cell lines known to express INCA1 (such as EL4 cells).

• Antibody concentration: Begin with 1-2 μg/mL concentration as demonstrated in validation studies .

• Secondary antibody selection: Use appropriate anti-rabbit IgG detection systems (HRP, AP, FITC, or biotin-conjugated secondary antibodies are compatible) .

• Controls: Include positive controls (cell lines with known INCA1 expression) and negative controls (INCA1 knockout/knockdown samples if available).

• Blocking: Standard blocking protocols with 5% non-fat milk or BSA in TBST should be sufficient.

Optimizing protein loading (20-50 μg total protein) and exposure times may be necessary depending on the level of INCA1 expression in your samples.

What experimental systems are appropriate for studying INCA1 function?

Based on published research, these experimental systems have proven valuable for INCA1 studies:

• Cell lines: EL4 cells have been used successfully for Western blot detection .

• Primary cells: Bone marrow samples can be used to study INCA1 expression in normal versus leukemic conditions .

• Animal models: Inca1 knockout mice are viable and fertile, making them excellent models for studying INCA1 function in vivo .

• Leukemia samples: INCA1 expression is significantly reduced in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patient samples compared to normal bone marrow .

When designing experiments, consider that INCA1 expression is influenced by cell cycle status - it is induced during cell cycle arrest and suppressed by mitogenic and oncogenic signals .

How does INCA1 regulate CDK activity and cell cycle progression?

INCA1 functions through a novel cyclin interaction domain that enables binding to CDK1- and CDK2-bound cyclins . This interaction inhibits CDK2 kinase activity, which has been demonstrated through kinase assays using recombinant proteins . Specifically:

• INCA1 binds directly to cyclins when they are in complex with CDK2

• This binding inhibits the catalytic activity of CDK2

• The inhibition of CDK2 activity leads to suppression of cell proliferation

In mouse embryonic fibroblasts (MEFs) lacking INCA1 (Inca1−/−), there is an increase in the fraction of S-phase cells, demonstrating INCA1's role in restricting S-phase entry . Additionally, INCA1 knockout mice display increased CDK2 activity in the spleen, correlated with altered spleen architecture .

What is the relationship between INCA1 and ING5 in growth regulation?

INCA1 serves as a critical co-factor for ING5 (Inhibitor of growth protein 5), a growth suppressor with reduced expression in AML . Key aspects of this relationship include:

• ING5 was identified as an interaction partner of INCA1 in a yeast-two-hybrid screen

• ING5's growth suppression function depends on its interaction with INCA1

• Both proteins show reduced expression in leukemic cells compared to normal bone marrow

• ING5 inhibits bone marrow colony formation, a function that requires INCA1

This interaction represents an important mechanism by which INCA1 contributes to growth regulation beyond its direct inhibition of CDK activity, linking INCA1 to broader cellular processes regulated by the ING family, including chromatin remodeling, apoptosis, and cell cycle control .

How is INCA1 expression altered in cancer and what are the implications?

INCA1 expression is significantly reduced in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patient samples compared to normal bone marrow . This reduced expression correlates with the growth-suppressive function of INCA1, suggesting that loss of INCA1 contributes to the unrestricted proliferation characteristic of leukemic cells.

The suppression of INCA1 by mitogenic and oncogenic signals provides a mechanistic explanation for its downregulation in cancer . The consequent increase in CDK2 activity would promote cell cycle progression and proliferation. Given these observations, INCA1 may function as a tumor suppressor in the context of leukemia.

Understanding the mechanisms of INCA1 downregulation in cancer could potentially reveal new therapeutic approaches. For instance, strategies to restore INCA1 expression or function might help to inhibit the proliferation of leukemic cells.

How can I validate INCA1 antibody specificity in my experimental system?

To ensure INCA1 antibody specificity:

• Genetic controls: Use INCA1 knockout or knockdown systems as negative controls. Inca1−/− mouse embryonic fibroblasts would be ideal negative controls .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to demonstrate binding specificity.

• Multiple antibodies: Use antibodies raised against different epitopes of INCA1 to confirm consistent detection patterns.

• Predicted molecular weight: Confirm that the detected band matches the expected molecular weight of INCA1 (~25 kDa, though this may vary with post-translational modifications).

• Expression pattern correlation: Compare antibody detection results with mRNA expression data from qRT-PCR using primers such as mINCA1-F301/mINCA1-R390 for mouse samples .

What experimental controls are critical for INCA1 functional studies?

When investigating INCA1 function, include these essential controls:

• Expression controls: Validate INCA1 overexpression or knockdown by Western blot and qRT-PCR.

• Cyclin-binding mutants: For studies of CDK inhibition, include mutants in the cyclin-binding domain as functional controls .

• Protein-protein interaction controls: For co-immunoprecipitation experiments, include appropriate IgG controls and antibody-only controls.

• Kinase assay controls: Include both positive controls (active CDK2/cyclin complexes) and negative controls (kinase inhibitors) when measuring the effect of INCA1 on CDK activity .

• Proliferation assay controls: Include standard growth-promoting and growth-inhibiting conditions when assessing INCA1's effect on proliferation.

What are promising research avenues involving INCA1 in cancer studies?

Several promising research directions emerge from current INCA1 knowledge:

• Therapeutic restoration: Investigating methods to restore INCA1 expression or function in leukemic cells might offer therapeutic benefit given its reduced expression in AML and ALL .

• Biomarker potential: Evaluating whether INCA1 expression levels correlate with disease progression or treatment response in leukemia patients.

• Pathway interactions: Further exploration of the INCA1-ING5 interaction and its implications for growth control in normal and malignant cells .

• Structural studies: Determining the three-dimensional structure of INCA1, particularly its cyclin-binding domain, could facilitate the design of mimetic compounds that replicate its CDK-inhibitory function.

• Regulatory mechanisms: Investigating the transcriptional and post-translational regulation of INCA1 to understand how its expression and function are controlled in normal and disease states.

How might INCA1 research integrate with broader cell cycle regulation studies?

INCA1 represents an interesting addition to the CDK inhibitor family. Unlike classic CDK inhibitors (p21, p27, p57, etc.), INCA1 knockout mice are viable and fertile , suggesting a more specialized or redundant function. Future research could explore:

• The specific contexts in which INCA1-mediated CDK inhibition is most critical

• Potential compensation mechanisms in Inca1−/− mice

• Interactions between INCA1 and other cell cycle regulators

• The evolutionary conservation and divergence of INCA1 function across species

This research could help place INCA1 in the broader context of cell cycle regulation and potentially reveal novel regulatory mechanisms.