TNPO2 Antibody

What is TNPO2 and what cellular functions does it perform?

Transportin 2 (TNPO2) is a nuclear transport receptor belonging to the importin-β superfamily. It functions primarily in nucleocytoplasmic transport of proteins containing M9 nuclear localization signals. TNPO2 has a calculated molecular weight of approximately 100 kDa (887 amino acids), though it typically appears between 90-100 kDa on western blots . The protein is encoded by the TNPO2 gene (NCBI Gene ID: 30000) and plays critical roles in both normal cellular function and disease states . Recent research indicates TNPO2 may function in cancer cell proliferation and inhibition of apoptosis, particularly in gastric cancer models .

What cellular and tissue systems express detectable levels of TNPO2?

TNPO2 expression has been detected across multiple experimental systems. Western blot analysis has confirmed TNPO2 expression in:

• Fetal human brain tissue

• HEK-293 cells

• HeLa cells

• Jurkat cells

• K-562 cells

Immunofluorescence has successfully detected TNPO2 in MCF-7 cells . The protein shows reactivity across human, mouse, and rat models, making it suitable for comparative studies across species .

What are the optimal antibody dilutions for different experimental applications?

Proper antibody dilution is essential for experimental success. Based on extensive validation, the following dilutions are recommended for TNPO2 antibody applications:

It is crucial to optimize these dilutions for your specific experimental system as reactivity may vary depending on sample type, detection method, and other experimental conditions .

How should TNPO2 antibodies be stored and handled to maintain reactivity?

To preserve antibody function and stability:

• Store TNPO2 antibodies at -20°C in the provided buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) .

• Antibodies are generally stable for 12 months from date of receipt when properly stored .

• Prepare small aliquots to avoid repeated freeze/thaw cycles that can compromise antibody integrity .

• For -20°C storage, aliquoting is considered unnecessary for some formulations, but it remains best practice to minimize freeze-thaw cycles .

• Note that some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer .

How can TNPO2 antibodies be employed to study its role in cancer progression?

Recent research has established TNPO2 as a potentially important factor in cancer biology. To investigate TNPO2's role in cancer:

• Proliferation studies: MTT assays can be used following TNPO2 siRNA knockdown or overexpression to assess effects on cell viability. This approach has revealed that TNPO2 promotes proliferation in gastric cancer cells .

• Cell cycle analysis: Flow cytometry with propidium iodide staining following TNPO2 manipulation can reveal effects on cell cycle progression. Specific protocol: Transfect cells with siTNPO2 for 48 hours, trypsinize, fix overnight with 75% alcohol (4°C), stain with 400 μL PBS containing 50 μg/mL PI and 100 μg/mL RNase at 37°C for 30 minutes, then analyze G1, S, and G2 phase distributions by flow cytometry .

• Regulatory relationship studies: RT-qPCR and Western blotting can establish relationships between TNPO2 and other signaling molecules. For example, research has identified DYNC1I1 as an upstream regulator of TNPO2 expression in gastric cancer cells .

What is the relationship between TNPO2 and transcription factors like SP1?

TNPO2 expression appears to be regulated by the transcription factor SP1. Studies have demonstrated:

• SP1 binds to the TNPO2 promoter region and upregulates its expression. This has been confirmed through luciferase reporter assays using pGL4.10-TNPO2 Promoter constructs (both wild-type and mutant) .

• The relationship between SP1 and TNPO2 has been verified through knockdown experiments, where silencing SP1 leads to significant downregulation of TNPO2 at both mRNA and protein levels .

• Computational prediction through the ALGGEN PROMO website and genecard website identified SP1 as a key transcription factor for TNPO2 .

• This regulatory relationship appears to be part of a larger signaling cascade involving DYNC1I1, which regulates SP1, which in turn regulates TNPO2 expression .

What is the recommended protocol for Western blot detection of TNPO2?

For optimal Western blot detection of TNPO2:

• Sample preparation: Total protein extraction using standard lysis buffers is suitable for most cell types. TNPO2 has been successfully detected in multiple cell lines including HEK-293, HeLa, Jurkat, and K-562 cells .

• Antibody selection: Use a validated anti-TNPO2 antibody at 1:500-1:2000 dilution . Multiple antibody options exist including rabbit polyclonal antibodies that have been affinity-purified against TNPO2 immunogens .

• Detection: TNPO2 typically appears at 90-100 kDa on Western blots, consistent with its calculated molecular weight of 100 kDa (887 amino acids) .

• Validation controls: Include positive control samples such as fetal human brain tissue or any of the validated cell lines mentioned above .

How can TNPO2 knockdown experiments be designed and validated?

To effectively knockdown TNPO2 and confirm the effect:

• siRNA design: Design siRNAs targeting TNPO2 mRNA. Published studies have successfully used siRNA approaches to silence TNPO2 expression in gastric cancer cell lines .

• Transfection: Transfect cells with siTNPO2 using standard transfection protocols appropriate for your cell type.

• Validation of knockdown efficiency:

  • RT-qPCR using primers specific to TNPO2 (e.g., F: 5′-CTTTAGGGTGTCTTCCGCGA-3′, R: 5′-GAAGACAGTAGGGCACCGTT-3′)

  • Western blotting with anti-TNPO2 antibody (recommended dilution 1:1000)

  • GAPDH can be used as an internal control for normalization in both techniques

• Functional assays: Following confirmed knockdown, assess functional consequences using appropriate assays:

  • Proliferation (MTT assay)

  • Apoptosis analysis

  • Cell cycle distribution (flow cytometry)

  • Expression analysis of potential downstream targets like P21

What are common challenges when using TNPO2 antibodies in immunofluorescence?

When performing immunofluorescence with TNPO2 antibodies:

• Optimal fixation: Standard 4% paraformaldehyde fixation is generally suitable, but optimization may be required depending on cell type.

• Antibody dilution: Start with the recommended range (1:300-1:1200 or 1:20-1:200 depending on antibody source) and optimize as needed . MCF-7 cells have been validated for IF/ICC applications of TNPO2 antibody .

• Background reduction: To minimize non-specific binding:

  • Include appropriate blocking steps

  • Optimize antibody concentration

  • Ensure sufficient washing between steps

  • Consider using highly purified antibody preparations (≥95% purity by SDS-PAGE)

• Control samples: Include positive controls (e.g., MCF-7 cells) and negative controls (primary antibody omission) to validate staining specificity .

How can co-immunoprecipitation experiments for TNPO2 interactions be optimized?

For successful co-immunoprecipitation (CoIP) of TNPO2 and its binding partners:

• Lysis conditions: Use appropriate lysis buffers that maintain protein-protein interactions while effectively solubilizing membrane components.

• Antibody selection: TNPO2 antibodies have been validated for CoIP applications . Use 2-5 μg of antibody per sample.

• Protocol optimization: A basic CoIP protocol includes:

  • Prepare cell lysates and quantify protein concentration

  • Pre-clear lysates with protein A/G beads

  • Incubate cleared lysates with TNPO2 antibody overnight at 4°C

  • Add protein A/G beads and incubate

  • Wash beads thoroughly (typically 4 times with lysis buffer)

  • Elute bound proteins by boiling in 40-50 μL of 2× sample buffer for 5 minutes

  • Analyze by Western blotting

• Controls: Include IgG isotype controls and input samples to validate specific interactions.