nifk Antibody
Description
Antibody Characteristics
NIFK antibodies are available in multiple formats with distinct validation profiles:
Key epitope regions:
Cancer Progression Mechanisms
NIFK promotes metastasis through:
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TCF/β-catenin pathway activation: Downregulates casein kinase 1α (CK1α) via RUNX1 repression, enhancing cell migration/invasion
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Ki-67 interaction: Binds Ki-67's FHA domain to drive proliferation (p<0.001 in A549/PC13 lung cancer cells)
In vivo data:
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NIFK overexpression increased lung metastasis nodules in NSG mice by 3.8-fold (p=0.0127)
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Silencing NIFK reduced tumor weight by 58% in xenograft models (p<0.01)
Clinical Correlations
| Cancer Type | Cohort Size | Survival Impact (High NIFK) | Key Association |
|---|---|---|---|
| Lung | 188 patients | 42% reduced 5-year survival | Lymph node metastasis (p=0.018) |
| Breast | 84 patients | 37% reduced DFS* | HER2+ subtypes |
*Disease-free survival
Technical Considerations
Optimal working concentrations:
Limitations:
Therapeutic Potential
While no NIFK-targeted therapies are clinically approved, research suggests:
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What is NIFK and why are antibodies against it important in research?
NIFK (MKI67 FHA Domain-Interacting Nucleolar Phosphoprotein) is a protein that plays crucial roles in cell cycle progression and ribosome biogenesis. It interacts with Ki-67 protein and participates in RNA processing pathways. NIFK antibodies are essential research tools for studying these cellular processes because they allow detection, quantification, and localization of NIFK in various experimental contexts .
What types of NIFK antibodies are available for research applications?
Several types of NIFK antibodies are available, including:
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Polyclonal antibodies that detect endogenous NIFK protein levels
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Antibodies targeting specific epitopes or phosphorylation sites (e.g., Tyr795, Ser234, Thr234)
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Antibodies recognizing specific amino acid sequences within NIFK
These antibodies vary in their host species (commonly rabbit), reactivity (human, mouse, etc.), and conjugation status. For instance, catalog ABIN3185906 is a rabbit polyclonal antibody targeting the Tyr795 site of human and mouse NIFK , while other antibodies might target different regions such as AA 1-293 or AA 232-260 .
What are the recommended applications and dilutions for NIFK antibodies?
NIFK antibodies can be used in multiple experimental applications with specific recommended dilutions:
| Application | Recommended Dilution | Notes |
|---|---|---|
| Western Blot (WB) | 1:1000-1:8000 | Detects ~34 kDa band in HeLa, HEK-293, HepG2 cells |
| Immunohistochemistry (IHC) | 1:200-1:2000 | Effective in human liver cancer and renal cell carcinoma tissues |
| Immunofluorescence (IF) | 1:50-1:500 | Validated in MCF-7 cells |
| ELISA | 1:5000 | For quantitative detection |
| Flow Cytometry (FC) | 0.40 μg per 10^6 cells | For intracellular detection |
These recommendations serve as starting points; optimal dilutions should be determined experimentally for each specific antibody and application .
How should sample preparation be optimized for NIFK antibody applications?
For optimal results with NIFK antibodies:
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For IHC applications, antigen retrieval with TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may also be effective
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For Western blot, standard protein extraction protocols work well, with NIFK typically observed at the expected molecular weight of 34 kDa
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For immunofluorescence, standard fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is generally effective
Cell types that reliably express NIFK include HeLa, HEK-293, and HepG2 cell lines, making them suitable positive controls for antibody validation .
How can NIFK antibodies be used to study ribosome biogenesis pathways?
NIFK plays a crucial role in ribosome biogenesis, specifically in rRNA maturation, through its RNA recognition motif (RRM). Researchers can use NIFK antibodies to:
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Visualize NIFK localization in nucleoli using immunofluorescence
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Assess NIFK protein levels in ribosome biogenesis studies via Western blot
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Investigate NIFK interactions with pre-rRNA processing machinery via immunoprecipitation
Studies have shown that NIFK depletion leads to accumulation of 45S/47S precursors and reduction of 30S/26S pre-rRNAs, indicating delayed processing at the ITS1 site 2. This results in impaired maturation of 28S and 5.8S rRNA .
Experimental design should include controls for RNA polymerase I inhibition (e.g., Actinomycin D) and analysis of 5S rRNA levels, as NIFK depletion affects the inverse correlation between 5S rRNA and p53 levels typically observed with other LSU biogenesis factors .
What is the role of NIFK in cancer progression and how can antibodies help investigate this?
NIFK promotes cancer progression through multiple mechanisms that can be investigated using NIFK antibodies:
What are common issues when working with NIFK antibodies and how can they be resolved?
| Issue | Possible Cause | Solution |
|---|---|---|
| Weak or no signal in Western blot | Insufficient protein, inadequate antibody concentration | Increase sample loading, optimize antibody dilution (try 1:1000 initially), extend incubation time |
| High background in immunostaining | Non-specific binding, inadequate blocking | Increase blocking time, optimize antibody dilution (1:100-1:300 for IHC), include additional washing steps |
| Inconsistent results between experiments | Antibody degradation, variation in sample preparation | Aliquot antibodies to avoid freeze-thaw cycles, standardize sample preparation protocols |
| Multiple bands in Western blot | Cross-reactivity, protein degradation | Verify with knockout or knockdown controls, add protease inhibitors during sample preparation |
| Nuclear versus nucleolar staining patterns | Different fixation methods affecting epitope accessibility | Compare 4% PFA versus methanol fixation, optimize permeabilization conditions |
When troubleshooting, it's advisable to validate NIFK knockdown or knockout samples as negative controls .
How can researchers validate the specificity of NIFK antibodies?
To ensure NIFK antibody specificity:
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Genetic validation: Use NIFK knockdown (via siRNA or shRNA) or knockout (via CRISPR-Cas9) samples as negative controls
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Western blot validation: Confirm the antibody detects a band of the expected molecular weight (34 kDa), though post-translational modifications may affect migration
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Cross-reactivity testing: Test the antibody against related proteins or in samples from multiple species if cross-species reactivity is claimed
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Multiple antibody approach: Use antibodies targeting different epitopes of NIFK to confirm specificity
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Immunoprecipitation followed by mass spectrometry: Confirm the identity of the protein being recognized
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Biophysics-informed modeling: Recent advances in computational analysis can help predict antibody specificity and cross-reactivity profiles
How are computational approaches enhancing NIFK antibody design and applications?
Recent advances in computational methods are transforming antibody research, including NIFK antibodies:
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Biophysics-informed modeling: This approach combines experimental data with computational analysis to predict and design antibodies with customized specificity profiles. Researchers can apply this to NIFK antibodies to:
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Structure-function prediction: Methods coupling structural data with repertoire information can identify antibodies with similar structural properties despite sequence differences. For NIFK antibody research, this could:
The position-specific structure-scoring matrix (P3SM) approach incorporating structure-prediction scores can identify antibodies with similar structural and functional properties, even with divergent sequences .
What emerging applications exist for studying NIFK-related RNA processing mechanisms?
NIFK's RNA recognition motif (RRM) is crucial for rRNA maturation, opening several research avenues:
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Pre-rRNA processing analysis: NIFK antibodies can help investigate how NIFK affects specific steps in pre-rRNA processing, particularly at the ITS1 site 2. Northern blot analysis with radiolabeled probes can detect accumulation of precursors (45S/47S) and reduction of intermediates (30S/26S)
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Protein-RNA interaction studies: RNA immunoprecipitation (RIP) using NIFK antibodies can identify the specific RNA sequences recognized by NIFK's RRM domain
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Nucleolar stress response: Investigation of NIFK's role in the nucleolar stress response pathway, particularly regarding p53 activation. NIFK depletion affects the relationship between 5S rRNA and p53 levels, suggesting complex regulatory mechanisms
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RRM domain mutagenesis studies: Antibodies recognizing different NIFK epitopes can track how specific mutations in the RRM domain affect NIFK localization and function
These approaches contribute to our understanding of ribosome biogenesis defects in cancer and other diseases, potentially identifying new therapeutic targets or diagnostic markers.
