Recombinant Human INKA2 Protein: Handling & Application Protocol

Recombinant Human INKA2 Protein is a research-grade reagent designed to support investigations into p53-mediated transcription, cell cycle arrest, and PAK4 kinase inhibition in mammalian systems.

Product Name

Recombinant Human INKA2 Protein

SKU

rp367336

Supplier Reference

absea#PP-2826

Protein Origin

Human (Homo sapiens)

Primary Research Area

p53 pathway, cell cycle regulation, PAK4 inhibition

Recommended Storage

-80 °C for long-term; avoid repeated freeze-thaw cycles

Typical Application

In vitro binding assays, kinase inhibition studies, Western blot, functional assays

Background and Biological Significance of INKA2

INKA2 (Inka Box Actin Regulator 2), also known as FAM212B, is a transcriptional target of the tumor suppressor p53. The INKA2 gene is induced upon p53 activation and encodes a protein that functions as a potent inhibitor of PAK4 (p21-activated kinase 4), a serine/threonine kinase involved in cytoskeletal reorganization, cell proliferation, and survival signaling.

Through PAK4 inhibition, INKA2 modulates actin dynamics and may contribute to p53-dependent cell cycle arrest and apoptosis. Its expression has been reported to be dysregulated in various cancer contexts, positioning INKA2 as a relevant target for oncology research. Recombinant Human INKA2 Protein enables researchers to study these molecular interactions in a controlled, cell-free or cell-based environment.

Reagent Preparation and Reconstitution

Upon receipt, briefly centrifuge the vial (e.g., 5–10 seconds at low speed) to collect any lyophilized material from the sides and cap before opening. Reconstitute the lyophilized Recombinant Human INKA2 Protein in sterile, nuclease-free water or an appropriate buffer (such as PBS containing 0.1% BSA or 5–50% glycerol) to a working concentration suitable for your assay.

General guideline: Reconstitute to a stock concentration of 0.1–1.0 mg/mL. Mix gently by pipetting; do not vortex. Allow the protein to dissolve for 10 minutes at room temperature, then transfer to ice. Prepare single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles, which can compromise protein activity and integrity.

Note: Specific reconstitution volume and recommended buffer conditions should be confirmed against the product datasheet for SKU rp367336, as formulation details may vary by lot.

Storage and Stability Recommendations

Proper storage is critical to maintaining the biological activity of Recombinant Human INKA2 Protein:

• Short-term (up to 1 week): Store at 4 °C if the protein will be used within several days of reconstitution.
• Long-term: Store lyophilized protein at -20 °C or -80 °C. After reconstitution, prepare aliquots and store at -80 °C for long-term preservation.
• Freeze-thaw cycles: Limit to a maximum of 2–3 cycles. Addition of a carrier protein (e.g., 0.1% BSA) or cryoprotectant (e.g., glycerol to 10–50% v/v) is recommended for aliquots stored at -80 °C.
• Avoid: Prolonged exposure to room temperature, repeated freeze-thaw cycles, and detergent contamination.

Note: Confirm specific stability data and recommended storage conditions from the official product datasheet for rp367336.

Experimental Applications and Assay Tips

Recombinant Human INKA2 Protein is suitable for several research applications:

• PAK4 Kinase Inhibition Assays: INKA2 can be used as an inhibitor of PAK4 activity in in vitro kinase assays. Titrate INKA2 concentration against a fixed amount of active PAK4 to determine IC₅₀ values. Include appropriate positive (kinase active) and negative (no kinase) controls.
• Protein–Protein Interaction Studies: Use in pull-down or co-immunoprecipitation setups to validate interactions between INKA2 and PAK4 or other candidate binding partners.
• Western Blot (as standard or control): Recombinant INKA2 can serve as a positive control or molecular weight marker in SDS-PAGE and Western blot experiments when using anti-INKA2 antibodies.
• Structural and Biophysical Studies: Suitable as input material for assays such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or thermal shift assays to characterize binding kinetics and thermodynamics.

Optimize protein concentrations empirically for each assay format. Pilot experiments using a concentration range (e.g., 0.1–10 µg/mL) are recommended before committing to large-scale experiments.

Quality Control and Troubleshooting

Quality Control: Recombinant Human INKA2 Protein (rp367336) is produced under controlled conditions. Typical quality control testing for recombinant proteins includes SDS-PAGE purity assessment and, where applicable, functional bioactivity testing. Refer to the Certificate of Analysis (CoA) provided with your specific lot for purity, concentration, and endotoxin data.

Common Troubleshooting Scenarios:

• Protein precipitation upon reconstitution: Try reconstituting at a lower concentration, increasing salt concentration in the buffer, or adding glycerol (5–10%). Ensure reconstitution buffer is at room temperature before addition.
• Low activity in kinase inhibition assay: Verify that the protein has not undergone excessive freeze-thaw cycles. Check that buffer conditions (pH, salt, reducing agents) are compatible with both INKA2 and PAK4 requirements.
• Unexpected band pattern on SDS-PAGE: Minor bands may represent protein degradation fragments or aggregates. Compare against the CoA gel image. If the pattern deviates significantly, contact Aladdin Scientific technical support.
• No signal in Western blot: Confirm antibody compatibility with recombinant INKA2 expressed in the chosen expression system. Adjust protein loading amount as needed.

Frequently asked questions

What is INKA2 and why is it relevant to cancer research?

INKA2 (FAM212B) is a p53-inducible protein that inhibits PAK4 kinase, a key regulator of cytoskeletal dynamics, cell proliferation, and survival. Because PAK4 is frequently overexpressed in tumors and p53 is the most commonly mutated tumor suppressor, understanding the INKA2–PAK4 axis offers insights into p53-dependent tumor suppression mechanisms. Recombinant INKA2 protein enables in vitro dissection of these interactions.

How should I reconstitute and store Recombinant Human INKA2 Protein (rp367336)?

Reconstitute the lyophilized protein in sterile water or a compatible buffer (e.g., PBS with 0.1% BSA) to a stock concentration of 0.1–1.0 mg/mL. Gently pipette to mix and allow 10 minutes at room temperature for complete dissolution. Immediately aliquot and store at -80 °C for long-term use. Avoid repeated freeze-thaw cycles to preserve activity. Always consult the product-specific CoA and datasheet for confirmed recommendations.

Which experimental applications is Recombinant Human INKA2 Protein suitable for?

Recombinant Human INKA2 Protein is suitable for in vitro PAK4 kinase inhibition assays, protein–protein interaction studies (pull-down, co-IP), Western blot positive controls, and biophysical characterization techniques such as SPR or ITC. Optimize protein concentrations for each application through pilot experiments, as optimal conditions vary by assay format and detection system.

What should I do if I observe protein precipitation after reconstitution?

If precipitation occurs, try reconstituting at a lower concentration, using a buffer with slightly higher ionic strength, or adding a cryoprotectant such as 5–10% glycerol. Ensure the buffer is at room temperature before adding to the lyophilized protein. If the issue persists, contact Aladdin Scientific technical support with your lot number for further guidance.

Is there a Certificate of Analysis available for this product?

Yes, a Certificate of Analysis (CoA) specific to each production lot of rp367336 is available. The CoA typically includes purity data from SDS-PAGE, protein concentration, and endotoxin levels. Contact Aladdin Scientific customer service or visit the product page to request the CoA for your specific lot number.

References

1. Barel O, et al. Studies on INKA2/FAM212B as a p53 transcriptional target and PAK4 inhibitor. [Details to be confirmed from primary literature]
2. Aladdin Scientific. Recombinant Human INKA2 Protein, SKU rp367336, Product Datasheet and Certificate of Analysis.
3. absea reference PP-2826. Technical documentation for Recombinant Human INKA2 Protein.

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